Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis

ABSTRACT

Compositions and methods are disclosed for stimulating or inhibiting angiogenesis and/or cardiovascularization in mammals, including humans. Pharmaceutical compositions are based on polypeptides or antagonists thereto that have been identified for one or more of these uses. Disorders that can be diagnosed, prevented, or treated by the compositions herein include trauma such as wounds, various cancers, and disorders of the vessels including atherosclerosis and cardiac hypertrophy.  
     In addition, the present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

1. FIELD OF THE INVENTION

[0001] The present invention relates to compositions and methods usefulfor the modulation (e.g., promotion or inhibition) of angiogenesisand/or cardiovascularization in mammals in need of such biologicaleffect. The present invention further relates to the diagnosis andtreatment of disorders involving angiogenesis (e.g., cardiovascular aswell as oncological disorders).

2. BACKGROUND OF THE INVENTION 2.1. Angiogenesis

[0002] Angiogenesis, defined as the growth or sprouting of new bloodvessels from existing vessels, is a complex process that primarilyoccurs during embryonic development. Under normal physiologicalconditions in adults, angiogenesis takes place only in very restrictedsituations such as hair growth and wounding healing (Auerbach, W. andAuerbach, R., 1994, Pharmacol Ther 63(3):265-311; Ribatti et al., 1991,Haematologica 76(4):311-20; Risau, 1997, Nature 386(6626):67 1-4).Unregulated angiogenesis has gradually been recognized to be responsiblefor a wide range of disorders, including, but not limited tocardiovascular disease, cancer, rheumatoid arthritis, psoriasis anddiabetic retinopathy (Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999,Circulation 99(13): 1653-5; Koch, 1998, Arthritis Rheum 41(6):951-62;Walsh, 1999, Rheumatology (Oxford) 38(2):103-12; Ware and Simons, 1997,Nat Med 3(2): 158-64).

2.2. Cardiac Disorders and Factors

[0003] Heart failure affects approximately five million Americans, andnew cases of heart failure number about 400,000 each year. It is thesingle most frequent cause of hospitalization for people age 65 andolder in the United States. Recent advances in the management of acutecardiac diseases, including acute myocardial infarction, are resultingin an expanding patient population that will eventually develop chronicheart failure. From 1979 to 1995, hospitalizations for congestive heartfailure (CHF) rose from 377,000 to 872,000 (a 130 percent increase) andCHF deaths increased 116 percent.

[0004] CHF is a syndrome characterized by left ventricular dysfunction,reduced exercise tolerance, impaired quality of life, and markedlyshortened life expectancy. The sine qua non of heart failure is aninability of the heart to pump blood at a rate sufficient to meet themetabolic needs of the body's tissues (in other words, there isinsufficient cardiac output).

[0005] At least four major compensatory mechanisms are activated in thesetting of heart failure to boost cardiac output, including peripheralvasoconstriction, increased heart rate, increased cardiac contractility,and increased plasma volume. These effects are mediated primarily by thesympathetic nervous system and the renin-angiotensin system. See,Eichhorn, American Journal of Medicine, 104: 163-169 (1998). Increasedoutput from the sympathetic nervous system increases vascular tone,heart rate, and contractility. Angiotensin II elevates blood pressureby 1) directly stimulating vascular smooth muscle contraction, 2)promoting plasma volume expansion by stimulating aldosterone andantidiuretic hormone secretion, 3) stimulating sympathetic-mediatedvascular tone, and 4) catalyzing the degradation of bradykinin, whichhas vasodilatory and natriuretic activity. See, review by Brown andVaughan, Circulation, 97: 1411-1420 (1998). As noted below, angiotensinII may also have directly deleterious effects on the heart by promotingmyocyte necrosis (impairing systolic function) and intracardiac fibrosis(impairing diastolic and in some cases systolic function). See, Weber,Circulation, 96: 4065-4082 (1998).

[0006] A consistent feature of congestive heart failure (CHF) is cardiachypertrophy, an enlargement of the heart that is activated by bothmechanical and hormonal stimuli and enables the heart to adapt todemands for increased cardiac output. Morgan and Baker, Circulation, 83:13-25 (1991). This hypertrophic response is frequently associated with avariety of distinct pathological conditions such as hypertension, aorticstenosis, myocardial infarction, cardiomyopathy, valvular regurgitation,and intracardiac shunt, all of which result in chronic hemodynamicoverload.

[0007] Hypertrophy is generally defined as an increase in size of anorgan or structure independent of natural growth that does not involvetumor formation. Hypertrophy of the heart is due either to an increasein the mass of the individual cells (myocytes), or to an increase in thenumber of cells making up the tissue (hyperplasia), or both. While theenlargement of an embryonic heart is largely dependent on an increase inmyocyte number (which continues until shortly after birth), post-natalcardiac myocytes lose their proliferative capacity. Further growthoccurs through hypertrophy of the individual cells.

[0008] Adult myocyte hypertrophy is initially beneficial as a short termresponse to impaired cardiac function by permitting a decrease in theload on individual muscle fibers. With severe, long-standing overload,however, the hypertrophied cells begin to deteriorate and die. Katz,“Heart Failure”, in: Katz A. M. ed., Physiology of the Heart (New York:Raven Press, 1992) pp. 638-668. Cardiac hypertrophy is a significantrisk factor for both mortality and morbidity in the clinical course ofheart failure. Katz, Trends Cardiovasc. Med., 5: 37-44 (1995). Forfurther details of the causes and pathology of cardiac hypertrophy see,e.g., Heart Disease, A Textbook of Cardiovascular Medicine, Braunwald,E. ed. (W. B. Saunders Co., 1988), Chapter 14, “Pathophysiology of HeartFailure.”

[0009] On a cellular level, the heart is composed of myocytes andsurrounding support cells, generically called non-myocytes. Whilenon-myocytes are primarily fibroblast/mesenchymal cells, they alsoinclude endothelial and smooth muscle cells. Indeed, although myocytesmake up most of the adult myocardial mass, they represent only about 30%of the total cell numbers present in heart. In response to hormonal,physiological, hemodynamic, and pathological stimuli, adult ventricularmuscle cells can adapt to increased workloads through the activation ofa hypertrophic process. This response is characterized by an increase inmyocyte cell size and contractile protein content of individual cardiacmuscle cells, without concomitant cell division and activation ofembryonic genes, including the gene for atrial natriuretic peptide(ANP). Chien et al., FASEB J., 5: 3037-3046 (1991); Chien et al., Annu.Rev. Physiol., 55: 77-95 (1993). An increment in myocardial massas aresult of an increase in myocyte size that is associated with anaccumulation of interstitial collagen within the extracellular matrixand around intramyocardial coronary arteries has been described in leftventricular hypertrophy secondary to pressure overload in humans.Caspari et al., Cardiovasc. Res., 11: 554-558 (1977); Schwarz et al.,Am. J. Cardiol., 42: 895-903 (1978); Hess et al., Circulation, 63:360-371 (1981); Pearlman et al., Lab. Invest., 46: 158-164 (1982).

[0010] It has also been suggested that paracrine factors produced bynon-myocyte supporting cells may additionally be involved in thedevelopment of cardiac hypertrophy, and various non-myocyte derivedhypertrophic factors, such as, leukocyte inhibitory factor (LIF) andendothelin, have been identified. Metcalf, Growth Factors, 7: 169-173(1992); Kurzrock et al., Endocrine Reviews, 12: 208-217 (1991); Inoue etal., Proc. Natl. Acad. Sci. USA, 86: 2863-2867 (1989); Yanagisawa andMasaki, Trends Pharm. Sci., 10: 374-378 (1989); U.S. Pat. No. 5,573,762(issued Nov. 12, 1996). Further exemplary factors that have beenidentified as potential mediators of cardiac hypertrophy includecardiotrophin-1 (CT-1) (Pennica et al., Proc. Nat. Acad. Sci. USA, 92:1142-1146 (1995)), catecholamines, adrenocorticosteroids, angiotensin,and prostaglandins.

[0011] At present, the treatment of cardiac hypertrophy varies dependingon the underlying cardiac disease. Catecholamines,adrenocorticosteroids, angiotensin, prostaglandins, LIF, endothelin(including endothelin-1, -2, and -3 and big endothelin), and CT-1 areamong the factors identified as potential mediators of hypertrophy. Forexample, beta-adrenergic receptor blocking drugs (beta-blockers, e.g.,propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol,penbutolol, acetobutolol, atenolol, metoprolol, carvedilol, etc.) andverapamil have been used extensively in the treatment of hypertrophiccardiomyopathy. The beneficial effects of beta-blockers on symptoms(e.g., chest pain) and exercise tolerance are largely due to a decreasein the heart rate with a consequent prolongation of diastole andincreased passive ventricular filling. Thompson et al., Br. Heart J.,44: 488-98 (1980); Harrison et al., Circulation, 29: 84-98 (1964).Verapamil has been described to improve ventricular filling and probablyreducing myocardial ischemia. Bonow et al., Circulation, 72: 853-64(1985).

[0012] Nifedipine and diltiazem have also been used occasionally in thetreatment of hypertrophic cardiomyopathy. Lorell et al., Circulation,65: 499-507 (1982); Betocchi et al., Am. J. Cardiol., 78: 451-457(1996). However, because of its potent vasodilating properties,nifedipine may be harmful, especially in patients with outflowobstruction. Disopyramide has been used to relieve symptoms by virtue ofits negative inotropic properties. Pollick, N. Engl. J. Med., 307:997-999 (1982). In many patients, however, the initial benefits decreasewith time. Wigle et al., Circulation, 92: 1680-1692 (1995).Antihypertensive drug therapy has been reported to have beneficialeffects on cardiac hypertrophy associated with elevated blood pressure.Examples of drugs used in antihypertensive therapy, alone or incombination, are calcium antagonists, e.g., nitrendipine; adrenergicreceptor blocking agents, e.g., those listed above; angiotensinconverting enzyme (ACE) inhibitors such as quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, and lisinopril; diuretics,e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, andindapamide; and calcium channel blockers, e g., diltiazem, nifedipine,verapamil, and nicardipine.

[0013] For example, treatment of hypertension with diltiazem andcaptopril showed a decrease in left ventricular muscle mass, but theDoppler indices of diastolic function did not normalize. Szlachcic etal., Am. J. Cardiol., 63: 198-201 (1989); Shahi et al., Lancet, 336:458-461 (1990). These findings were interpreted to indicate thatexcessive amounts of interstitial collagen may remain after regressionof left ventricular hypertrophy. Rossi et al., Am. Heart J., 124:700-709 (1992). Rossi et al., supra, investigated the effect ofcaptopril on the prevention and regression of myocardial cellhypertrophy and interstitial fibrosis in pressure overload cardiachypertrophy, in experimental rats.

[0014] Agents that increase cardiac contractility directly (iontropicagents) were initially thought to benefit patients with heart failurebecause they improved cardiac output in the short term. However, allpositive inotropic agents except digoxigenin have been found to resultin increased long-term mortality, in spite of short-term improvements incardiac performance. Massie, Curr. Op. in Cardiology, 12: 209-217(1997); Reddy et al., Curr. Opin. Cardiol., 12: 233-241 (1997).Beta-adrenergic receptorblockers have recently been advocated for use inheart failure. Evidence from clinical trials suggests that improvementsin cardiac function can be achieved without increased mortality, thoughdocumented improvements of patient survival have not yet beendemonstrated. See also, U.S. Pat. Nos. 5,935,924, 5,624,806; 5,661,122;and 5,610,134 and WO 95/28173 regarding the use of cardiotropin-1 orantagonists thereof, or growth hormone and/or insulin-like growthfactor-I in the treatment of CHF. Another treatment modality is hearttransplantation, but this is limited by the availability of donorhearts.

[0015] Endothelin is a vasoconstricting peptide comprising 21 aminoacids, isolated from swine arterial endothelial culture supernatant andstructurally determined. Yanagisawa et al., Nature, 332: 411-415 (1988).Endothelin was later found to exhibit various actions, and endothelinantibodies as endothelin antagonists have proven effective in thetreatment of myocardial infarction, renal failure, and other diseases.Since endothelin is present in live bodies and exhibits vasoconstrictingaction, it is expected to be an endogenous factor involved in theregulation of the circulatory system, and may be associated withhypertension, cardiovascular diseases such as myocardial infarction, andrenal diseases such as acute renal failure. Endothelin antagonists aredescribed, for example, in U.S. Pat. No. 5,773,414; JP Pat. Publ.3130299/1991, EP 457,195; EP 460,679; and EP 552,489. A new endothelin Breceptor for identifying endothelin receptor antagonists is described inU.S. Pat. No. 5,773,223.

[0016] Current therapy for heart failure is primarily directed to usingangiotensin-converting enzyme (ACE) inhibitors, such as captopril, anddiuretics. These drugs improve hemodynamic profile and exercisetolerance and reduce the incidence of morbidity and mortality inpatients with CHF. Kramer et al., Circulation, 67(4): 807-816 (1983);Captopril Multicenter Research Group, J.A.C.C., 2(4): 755-763 (1983);The CONSENSUS Trial Study Group, N. Engl. J. Med., 316(23): 1429-1435(1987); The SOLVD Investigators, N. Engl. J. Med., 325(5): 293-302(1991). Further, they are useful in treating hypertension, leftventricular dysfunction, atherosclerotic vascular disease, and diabeticnephropathy. Brown and Vaughan, supra. However, despite proven efficacy,response to ACE inhibitors has been limited. For example, whileprolonging survival in the setting of heart failure, ACE inhibitorsappear to slow the progression towards end-stage heart failure, andsubstantial numbers of patients on ACE inhibitors have functional classIII heart failure.

[0017] Moreover, improvement of functional capacity and exercise time isonly small and mortality, although reduced, continues to be high. TheCONSENSUS Trial Study Group, N. Engl. J. Med., 316(23): 1429-1453(1987); The SOLVD Investigators, N. Engl. J. Med., 325(5): 293-302(1991); Cohn et al., N. Engl. J. Med., 325(5): 303-310 (1991); TheCaptopril-Digoxin Multicenter Research Group, JAMA, 259(4): 539-544(1988). Hence, ACE inhibitors consistently appear unable to relievesymptoms in more than 60% of heart failure patients and reduce mortalityof heart failure only by approximately 15-20%. For further adverseeffects, see Brown and Vaughan, supra.

[0018] An alternative to ACE inhibitors is represented by specific AT1receptor antagonists. Clinical studies are planned to compare theefficacy of these two modalities in the treatment of cardiovascular andrenal disease. However, animal model data suggests that the ACE/Ang IIpathway, while clearly involved in cardiac hypertrophy, is not the only,or even the primary pathway active in this role. Mouse genetic“knockout” models have been made to test individual components of thepathway. In one such model, the primary cardiac receptor for Ang II, ATsub 1A, has been genetically deleted; these mice do not develophypertrophy when Ang II is given experimentally (confirming the basicsuccess of the model in eliminating hypertrophy secondary to Ang II).However, when the aorta is constricted in these animals (a model ofhypertensive cardiac stress), the hearts still become hypertrophic. Thissuggests that alternative signaling pathways, not depending on thisreceptor (AT sub 1A), are activated in hypertension. ACE inhibitorswould presumably not be able to inhibit these pathways. See, Harada etal., Circulation, 97: 1952-1959 (1998). See also, Homcy, Circulation,97: 1890-1892 (1998) regarding the enigma associated with the processand mechanism of cardiac hypertrophy.

[0019] About 750,000 patients suffer from acute myocardial infarction(AMI) annually, and approximately one-fourth of all deaths in the UnitedStates are due to AMI. In recent years, thrombolytic agents, e.g.,streptokinase, urokinase, and in particular tissue plasminogen activator(t-PA) have significantly increased the survival of patients whosuffered myocardial infarction. When administered as a continuousintravenous infusion over 1.5 to 4 hours, t-PA produces coronary patencyat 90 minutes in 69% to 90% of the treated patients. Topol et al., Am.J. Cardiol., 61: 723-728 (1988); Neuhaus et al., J. Am. Coll. Cardiol.,12: 581-587 (1988); Neuhaus et al., J. Am. Coll. Cardiol., 14: 1566-1569(1989). The highest patency rates have been reported with high dose oraccelerated dosing regimens. Topol, J. Am. Coll. Cardiol., 15: 922-924(1990). t-PA may also be administered as a single bolus, although due toits relatively short half-life, it is better suited for infusiontherapy. Tebbe et al., Am. J. Cardiol., 64: 448-453 (1989). A t-PAvariant, specifically designed to have longer half-life and very highfibrin specificity, TNK t-PA (a T103N, N117Q, KHRR(296-299)AAAA t-PAvariant, Keyt et al., Proc. Natl. Acad. Sci. USA, 91: 3670-3674 (1994))is particularly suitable for bolus administration. However, despite allthese advances, the long-term prognosis of patient survival dependsgreatly on the post-infarction monitoring and treatment of the patients,which should include monitoring and treatment of cardiac hypertrophy.

2.3. Growth Factors

[0020] Various naturally occurring polypeptides reportedly induce theproliferation of endothelial cells. Among those polypeptides are thebasic and acidic fibroblast growth factors (FGF) (Burgess and Maciag,Annual Rev. Biochem., 58: 575 (1989)), platelet-derived endothelial cellgrowth factor (PD-ECGF) (Ishikawa et al., Nature, 338: 557 (1989)), andvascular endothelial growth factor (VEGF). Leung et al., Science, 246:1306 (1989); Ferrara and Henzel, Biochem. Biophys. Res. Commun., 161:851 (1989); Tischer et al., Biochem. Biophys. Res. Commun., 165: 1198(1989); EP 471,754B granted Jul. 31, 1996.

[0021] Media conditioned by cells transfected with the human VEGF(hVEGF) cDNA promoted the proliferation of capillary endothelial cells,whereas control cells did not. Leung et al., Science, 246: 1306 (1989).Several additional cDNAs were identified in human cDNA libraries thatencode 121-, 189-, and 206-amino acid isoforms of hVEGF (alsocollectively referred to as hVEGF-related proteins). The 121-amino acidprotein differs from hVEGF by virtue of the deletion of the 44 aminoacids between residues 116 and 159 in hVEGF. The 189-amino acid proteindiffers from hVEGF by virtue of the insertion of 24 amino acids atresidue 116 in hVEGF, and apparently is identical to human vascularpermeability factor (hVPF). The 206-amino acid protein differs fromhVEGF by virtue of an insertion of 41 amino acids at residue 116 inhVEGF. Houck et al., Mol. Endocrin., 5: 1806 (1991); Ferrara et al., J.Cell. Biochem., 47: 211 (1991); Ferrara et al., Endocrine Reviews, 13:18 (1992); Keck et al., Science, 246: 1309 (1989); Connolly et al., J.Biol. Chem., 264: 20017 (1989); EP 370,989 published May 30, 1990.

[0022] It is now well established that angiogenesis, which involves theformation of new blood vessels from preexisting endothelium, isimplicated in the pathogenesis of a variety of disorders. These includesolid tumors and metastasis, atherosclerosis, retrolental fibroplasia,hemangiomas, chronic inflammation, intraocular neovascular syndromessuch as proliferative retinopathies, e.g., diabetic retinopathy,age-related macular degeneration (AMD), neovascular glaucoma, immunerejection of transplanted corneal tissue and other tissues, rheumatoidarthritis, and psoriasis. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992); Klagsbrun et al., Annu. Rev. Physiol., 53: 217-239(1991); and Garner A., “Vascular diseases”, In: Pathobiology of OcularDisease. A Dynamic Approach, Garner A., Klintworth G K, eds., 2ndEdition (Marcel Dekker, NY, 1994), pp 1625-1710.

[0023] In the case of tumor growth, angiogenesis appears to be crucialfor the transition from hyperplasia to neoplasia, and for providingnourishment for the growth and metastasis of the tumor. Folkman et al.,Nature, 339: 58 (1989). The neovascularization allows the tumor cells toacquire a growth advantage and proliferative autonomy compared to thenormal cells. A tumor usually begins as a single aberrant cell which canproliferate only to a size of a few cubic millimeters due to thedistance from available capillary beds, and it can stay ‘dormant’without further growth and dissemination for a long period of time. Sometumor cells then switch to the angiogenic phenotype to activateendothelial cells, which proliferate and mature into new capillary bloodvessels. These newly formed blood vessels not only allow for continuedgrowth of the primary tumor, but also for the dissemination andrecolonization of metastatic tumor cells. Accordingly, a correlation hasbeen observed between density of microvessels in tumor sections andpatient survival in breast cancer as well as in several other tumors.Weidner et al., N. Engl. J. Med, 324: 1-6 (1991); Horak et a., Lancet,340: 1120-1124 (1992); Macchiarini et al., Lancet, 340: 145-146 (1992).The precise mechanisms that control the angiogenic switch is not wellunderstood, but it is believed that neovascularization of tumor massresults from the net balance of a multitude of angiogenesis stimulatorsand inhibitors (Folkman, 1995, Nat Med 1(1):27-31).

[0024] The search for positive regulators of angiogenesis has yieldedmany candidates, including aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α,angiogenin, IL-8, etc. Folkman et al., J.B.C., supra, and Klagsbrun etal., supra. The negative regulators so far identified includethrombospondin (Good et al., Proc. Natl. Acad. Sci. USA., 87: 6624-6628(1990)), the 16-kilodalton N-terminal fragment of prolactin (Clapp etal., Endocrinology, 133: 1292-1299 (1993)), angiostatin (O'Reilly etal., Cell, 79: 315-328 (1994)), and endostatin. O'Reilly et al., Cell,88: 277-285 (1996).

[0025] Work done over the last several years has established the keyrole of VEGF, not only in stimulating vascular endothelial cellproliferation, but also in inducing vascular permeability andangiogenesis. Ferrara et al., Endocr. Rev., 18: 4-25 (1997). The findingthat the loss of even a single VEGF allele results in embryoniclethality points to an irreplaceable role played by this factor in thedevelopment and differentiation of the vascular system. Furthermore,VEGF has been shown to be a key mediator of neovascularizationassociated with tumors and intraocular disorders. Ferrara et al.,Endocr. Rev., supra. The VEGF mRNA is overexpressed by the majority ofhuman tumors examined. Berkman et al., J. Clin. Invest., 91: 153-159(1993); Brown et al., Human Pathol., 26: 86-91 (1995); Brown et al.,Cancer Res., 53: 4727-4735 (1993); Mattern et al., Brit. J. Cancer, 73:931-934 (1996) Dvorak et al., Am. J. Pathol., 146: 1029-1039 (1995).

[0026] Also, the concentration levels of VEGF in eye fluids are highlycorrelated to the presence of active proliferation of blood vessels inpatients with diabetic and other ischemia-related retinopathies. Aielloet al., N Engl. J. Med., 331: 1480-1487 (1994). Furthermore, recentstudies have demonstrated the localization of VEGF in choroidalneovascular membranes in patients affected by AMD. Lopez et al., Invest.Ophthalmol. Vis. Sci., 37: 855-868 (1996).

[0027] Anti-VEGF neutralizing antibodies suppress the growth of avariety of human tumor cell lines in nude mice (Kim et al., Nature, 362:841-844 (1993); Warren et al., J. Clin. Invest., 95: 1789-1797 (1995);Borgstrom et al., Cancer Res., 56: 4032-4039 (1996); Melnyk et al.,Cancer Res., 56: 921-924 (1996)) and also inhibit intraocularangiogenesis in models of ischemic retinal disorders. Adamis et al.,Arch. Ophthalmol., 114: 66-71 (1996). Therefore, anti-VEGF monoclonalantibodies or other inhibitors of VEGF action are promising candidatesfor the treatment of solid tumors and various intraocular neovasculardisorders. Such antibodies are described, for example, in EP 817,648published Jan. 14, 1998 and in WO98/45331 and WO98/45332 both publishedOct. 15, 1998.

[0028] There exist several other growth factors and mitogens, includingtransforming oncogenes, that are capable of rapidly inducing a complexset of genes to be expressed by certain cells. Lau and Nathans,Molecular Aspects of Cellular Regulation, 6: 165-202 (1991). Thesegenes, which have been named immediate-early- or early-response genes,are transcriptionally activated within minutes after contact with agrowth factor or mitogen, independent of de novo protein synthesis. Agroup of these intermediate-early genes encodes secreted, extracellularproteins that are needed for coordination of complex biologicalprocesses such as differentiation and proliferation, regeneration, andwound healing. Ryseck et al., Cell Growth Differ., 2: 235-233 (1991).

[0029] Highly-related proteins that belong to this group include cef 10(Simmons et al., Proc. Natl. Acad. Sci. USA, 86:1178-1182 (1989)), cyr61, which is rapidly activated by serum- or platelet-derived growthfactor(PDGF (O'Brien et al., Mol. Cell Biol., 10: 3569-3577 (1990),human connective tissue growth factor (CTGF) (Bradham et al., J. Cell.Biol., 114: 1285-1294 (1991)), which is secreted by human vascularendothelial cells in high levels after activation with transforminggrowth factor beta (TGF-β), exhibits PDGF-like biological andimmunological activities, and competes with PDGF for a particular cellsurface receptor,fisp-12 (Ryseck et al., Cell Growth Differ., 2: 235-233(1991)), human vascular IBP-like growth factor (VIGF) (WO 96/17931), andnov, normally arrested in adult kidney cells, which was found to beoverexpressed in myeloblastosis-associated-virus-type-1-inducednephroblastomas. Joloit et al., Mol. Cell. Biol., 12: 10-21 (1992).

[0030] The expression of these immediate-early genes acts as “thirdmessengers” in the cascade of events triggered by growth factors. It isalso thought that they are needed to integrate and coordinate complexbiological processes, such as differentiation and wound healing in whichcell proliferation is a common event.

[0031] As additional mitogens, insulin-like growth factor bindingproteins (IGFBPs) have been shown, in complex with insulin-like growthfactor (IGF), to stimulate increased binding of IGF to fibroblast andsmooth muscle cell surface receptors. Clemmons et al., J. Clin. Invest.,77: 1548 (1986). Inhibitory effects of IGFBP on various IGF actions invitro include stimulation of glucose transport by adipocytes, sulfateincorporation by chondrocytes, and thymidine incorporation infibroblast. Zapf e al., J. Clin. Invest., 63: 1077 (1979). In addition,inhibitory effects of IGFBPs on growth factor-mediated mitogen activityin normal cells have been shown.

2.4. Need for Further Treatments

[0032] In view of the role of vascular endothelial cell growth andangiogenesis in many diseases and disorders, it is desirable to have ameans of reducing or inhibiting one or more of the biological effectscausing these processes. It is also desirable to have a means ofassaying for the presence of pathogenic polypeptides in normal anddiseased conditions, and especially cancer. Further, in a specificaspect, as there is no generally applicable therapy for the treatment ofcardiac hypertrophy, the identification of factors that can prevent orreduce cardiac myocyte hypertrophy is of primary importance in thedevelopment of new therapeutic strategies to inhibit pathophysiologicalcardiac growth. While there are several treatment modalities for variouscardiovascular and oncologic disorders, there is still a need foradditional therapeutic approaches.

3. SUMMARY OF THE INVENTION

[0033] The present invention provides compositions and methods formodulating (e.g., promoting or inhibiting) angiogenesis and/orcardiovascularization in mammals. The present invention is based on theidentification of compounds (i.e., proteins) that test positive invarious cardiovascular assays that test modulation (e.g., promotion orinhibition) of certain biological activities. Accordingly, the compoundsare believed to be useful drugs and/or drug components for the diagnosisand/or treatment (including prevention and amelioration) of disorderswhere such effects are desired, such as the promotion or inhibition ofangiogenesis, inhibition or stimulation of vascular endothelial cellgrowth, stimulation of growth or proliferation of vascular endothelialcells, inhibition of tumor growth, inhibition of angiogenesis-dependenttissue growth, stimulation of angiogenesis-dependent tissue growth,inhibition of cardiac hypertrophy and stimulation of cardiachypertrophy, e.g., for the treatment of congestive heart failure. Inaddition, the compositions and methods of the invention provide for thediagnostic monitoring of patients undergoing clinical evaluation for thetreatment of angiogenesis-related disorders, for monitoring the efficacyof compounds in clinical trials and for identifying subjects who may bepredisposed to such angiogenic-related disorders.

[0034] In one embodiment, the present invention provides a compositioncomprising a PRO polypeptide, an agonist or antagonist thereof, or ananti-PRO antibody in admixture with a pharmaceutically acceptablecarrier. In one aspect, the composition comprises a therapeuticallyeffective amount of the polypeptide, agonist, antagonist or antibody. Inanother aspect, the composition comprises a further active ingredient,namely, a cardiovascular, endothelial or angiogenic agent or anangiostatic agent, preferably an angiogenic or angiostatic agent.Preferably, the composition is sterile. The PRO polypeptide, agonist,antagonist or antibody may be administered in the form of a liquidpharmaceutical formulation, which may be preserved to achieve extendedstorage stability. Preserved liquid pharmaceutical formulations mightcontain multiple doses of PRO polypeptide, agonist, antagonist orantibody, and might, therefore, be suitable for repeated use. In apreferred embodiment, where the composition comprises an antibody, theantibody is a monoclonal antibody, an antibody fragment, a humanizedantibody, or a single-chain antibody.

[0035] In a further embodiment, the present invention provides a methodfor preparing such a composition useful for the treatment of acardiovascular, endothelial or angiogenic disorder comprising admixing atherapeutically effective amount of a PRO polypeptide, agonist,antagonist or antibody with a pharmaceutically acceptable carrier.

[0036] In a still further aspect, the present invention provides anarticle of manufacture comprising:

[0037] (a) a composition of matter comprising a PRO polypeptide oragonist or antagonist thereof;

[0038] (b) a container containing said composition; and

[0039] (c) a label affixed to said container, or a package insertincluded in said container referring to the use of said PRO polypeptideor agonist or antagonist thereof in the treatment of a cardiovascular,endothelial or angiogenic disorder, wherein the agonist or antagonistmay be an antibody which binds to the PRO polypeptide. The compositionmay comprise a therapeutically effective amount of the PRO polypeptideor the agonist or antagonist thereof.

[0040] In another embodiment, the present invention provides a methodfor identifying an agonist of a PRO polypeptide comprising:

[0041] (a) contacting cells and a test compound to be screened underconditions suitable for the induction of a cellular response normallyinduced by a PRO polypeptide; and

[0042] (b) determining the induction of said cellular response todetermine if the test compound is an effective agonist, wherein theinduction of said cellular response is indicative of said test compoundbeing an effective agonist.

[0043] In another embodiment, the present invention provides a methodfor identifying an agonist of a PRO polypeptide comprising:

[0044] (a) contacting cells and a test compound to be screened underconditions suitable for the stimulation of cell proliferation by a PROpolypeptide; and

[0045] (b) measuring the proliferation of said cells to determine if thetest compound is an effective agonist, wherein the stimulation of cellproliferation is indicative of said test compound being an effectiveagonist.

[0046] In another embodiment, the invention provides a method foridentifying a compound that inhibits the activity of a PRO polypeptidecomprising contacting a test compound with a PRO polypeptide underconditions and for a time sufficient to allow the test compound andpolypeptide to interact and determining whether the activity of the PROpolypeptide is inhibited. In a specific preferred aspect, either thetest compound or the PRO polypeptide is immobilized on a solid support.In another preferred aspect, the non-immobilized component carries adetectable label. In a preferred aspect, this method comprises the stepsof:

[0047] (a) contacting cells and a test compound to be screened in thepresence of a PRO polypeptide under conditions suitable for theinduction of a cellular response normally induced by a PRO polypeptide;and

[0048] (b) determining the induction of said cellular response todetermine if the test compound is an effective antagonist.

[0049] In another preferred aspect, this process comprises the steps of:

[0050] (a) contacting cells and a test compound to be screened in thepresence of a PRO polypeptide under conditions suitable for thestimulation of cell proliferation by a PRO polypeptide; and

[0051] (b) measuring the proliferation of the cells to determine if thetest compound is an effective antagonist.

[0052] In another embodiment, the invention provides a method foridentifying a compound that inhibits the expression of a PRO polypeptidein cells that normally expresses the polypeptide, wherein the methodcomprises contacting the cells with a test compound and determiningwhether the expression of the PRO polypeptide is inhibited. In apreferred aspect, this method comprises the steps of:

[0053] (a) contacting cells and a test compound to be screened underconditions suitable for allowing expression of the PRO polypeptide; and

[0054] (b) determining the inhibition of expression of said polypeptide.

[0055] In a still further embodiment, the invention provides a compoundthat inhibits the expression of a PRO polypeptide, such as a compoundthat is identified by the methods set forth above.

[0056] Another aspect of the present invention is directed to an agonistor an antagonist of a PRO polypeptide which may optionally be identifiedby the methods described above.

[0057] One type of antagonist of a PRO polypeptide that inhibits one ormore of the functions or activities of the PRO polypeptide is anantibody. Hence, in another aspect, the invention provides an isolatedantibody that binds a PRO polypeptide. In a preferred aspect, theantibody is a monoclonal antibody, which preferably has non-humancomplementarity-determining-region (CDR) residues and humanframework-region (FR) residues. The antibody may be labeled and may beimmobilized on a solid support. In a further aspect, the antibody is anantibody fragment, a single-chain antibody, or a humanized antibody.Preferably, the antibody specifically binds to the polypeptide.

[0058] In a still further aspect, the present invention provides amethod for diagnosing a disease or susceptibility to a disease which isrelated to a mutation in a PRO polypeptide-encoding nucleic acidsequence comprising determining the presence or absence of said mutationin the PRO polypeptide nucleic acid sequence, wherein the presence orabsence of said mutation is indicative of the presence of said diseaseor susceptibility to said disease.

[0059] In a still further aspect, the invention provides a method ofdiagnosing a cardiovascular, endothelial or angiogenic disorder in amammal which comprises analyzing the level of expression of a geneencoding a PRO polypeptide (a) in a test sample of tissue cells obtainedfrom said mammal, and (b) in a control sample of known normal tissuecells of the same cell type, wherein a higher or lower expression levelin the test sample as compared to the control sample is indicative ofthe presence of a cardiovascular, endothelial or angiogenic disorder insaid mammal. The expression of a gene encoding a PRO polypeptide mayoptionally be accomplished by measuring the level of mRNA or thepolypeptide in the test sample as compared to the control sample.

[0060] In a still further aspect, the present invention provides amethod of diagnosing a cardiovascular, endothelial or angiogenicdisorder in a mammal which comprises detecting the presence or absenceof a PRO polypeptide in a test sample of tissue cells obtained from saidmammal, wherein the presence or absence of said PRO polypeptide in saidtest sample is indicative of the presence of a cardiovascular,endothelial or angiogenic disorder in said mammal.

[0061] In a still further embodiment, the invention provides a method ofdiagnosing a cardiovascular, endothelial or angiogenic disorder in amammal comprising (a) contacting an anti-PRO antibody with a test sampleof tissue cells obtained from the mammal, and (b) detecting theformation of a complex between the antibody and the PRO polypeptide inthe test sample, wherein the formation of said complex is indicative ofthe presence of a cardiovascular, endothelial or angiogenic disorder inthe mammal. The detection may be qualitative or quantitative, and may beperformed in comparison with monitoring the complex formation in acontrol sample of known normal tissue cells of the same cell type. Alarger or smaller quantity of complexes formed in the test sampleindicates the presence of a cardiovascular, endothelial or angiogenicdysfunction in the mammal from which the test tissue cells wereobtained. The antibody preferably carries a detectable label. Complexformation can be monitored, for example, by light microscopy, flowcytometry, fluorimetry, or other techniques known in the art. The testsample is usually obtained from an individual suspected to have acardiovascular, endothelial or angiogenic disorder.

[0062] In another embodiment, the invention provides a method fordetermining the presence of a PRO polypeptide in a sample comprisingexposing a sample suspected of containing the PRO polypeptide to ananti-PRO antibody and determining binding of said antibody to acomponent of said sample. In a specific aspect, the sample comprises acell suspected of containing the PRO polypeptide and the antibody bindsto the cell. The antibody is preferably detectably labeled and/or boundto a solid support.

[0063] In further aspects, the invention provides a cardiovascular,endothelial or angiogenic disorder diagnostic kit comprising an anti-PROantibody and a carrier in suitable packaging. Preferably, such kitfurther comprises instructions for using said antibody to detect thepresence of the PRO polypeptide. Preferably, the carrier is a buffer,for example. Preferably, the cardiovascular, endothelial or angiogenicdisorder is cancer.

[0064] In yet another embodiment, the present invention provides amethod for treating a cardiovascular, endothelial or angiogenic disorderin a mammal comprising administering to the mammal an effective amountof a PRO polypeptide. Preferably, the disorder is cardiac hypertrophy,trauma such as wounds or burns, or a type of cancer. In a furtheraspect, the mammal is further exposed to angioplasty or a drug thattreats cardiovascular, endothelial or angiogenic disorders such as ACEinhibitors or chemotherapeutic agents if the cardiovascular, endothelialor angiogenic disorder is a type of cancer. Preferably, the mammal ishuman, preferably one who is at risk of developing cardiac hypertrophyand more preferably has suffered myocardial infarction.

[0065] In another preferred aspect, the cardiac hypertrophy ischaracterized by the presence of an elevated level of PGF_(2α).Alternatively, the cardiac hypertrophy may be induced by myocardialinfarction, wherein preferably the administration of the PRO polypeptideis initiated within 48 hours, more preferably within 24 hours, followingmyocardial infarction.

[0066] In another preferred embodiment, the cardiovascular, endothelialor angiogenic disorder is cardiac hypertrophy and said PRO polypeptideis administered together with a cardiovascular, endothelial orangiogenic agent. The preferred cardiovascular, endothelial orangiogenic agent for this purpose is selected from the group consistingof an antihypertensive drug, an ACE inhibitor, an endothelin receptorantagonist and a thrombolytic agent. If a thrombolytic agent isadministered, preferably the PRO polypeptide is administered followingadministration of such agent. More preferably, the thrombolytic agent isrecombinant human tissue plasminogen activator.

[0067] In another preferred aspect, the cardiovascular, endothelial orangiogenic disorder is cardiac hypertrophy and the PRO polypeptide isadministered following primary angioplasty for the treatment of acutemyocardial infarction, preferably wherein the mammal is further exposedto angioplasty or a cardiovascular, endothelial, or angiogenic agent.

[0068] In another preferred embodiment, the cardiovascular, endothelialor angiogenic disorder is a cancer and the PRO polypeptide isadministered in combination with a chemotherapeutic agent, a growthinhibitory agent or a cytotoxic agent.

[0069] In a further embodiment, the invention provides a method fortreating a cardiovascular, endothelial or angiogenic disorder in amammal comprising administering to the mammal an effective amount of aPRO polypeptide agonist, antagonist or anti-PRO antibody. Preferably,the cardiovascular, endothelial or angiogenic disorder is cardiachypertrophy, trauma, a cancer, or age-related macular degeneration. Alsopreferred is where the mammal is human, and where an effective amount ofan angiogenic or angiostatic agent is administered in conjunction withthe agonist, antagonist or anti-PRO antibody.

[0070] In still further embodiments, the invention provides a method fortreating a cardiovascular, endothelial or angiogenic disorder in amammal that suffers therefrom comprising administering to the mammal anucleic acid molecule that codes for either (a) a PRO polypeptide, (b)an agonist of a PRO polypeptide or (c) an antagonist of a PROpolypeptide, wherein said agonist or antagonist may be an anti-PROantibody. In a preferred embodiment, the mammal is human. In anotherpreferred embodiment, the gene is administered via ex vivo gene therapy.In a further preferred embodiment, the gene is comprised within avector, more preferably an adenoviral, adeno-associated viral,lentiviral, or retroviral vector.

[0071] In yet another aspect, the invention provides a recombinantretroviral particle comprising a retroviral vector consistingessentially of a promoter, nucleic acid encoding (a) a PRO polypeptide,(b) an agonist polypeptide of a PRO polypeptide, or (c) an antagonistpolypeptide of a PRO polypeptide, and a signal sequence for cellularsecretion of the polypeptide, wherein the retroviral vector is inassociation with retroviral structural proteins. Preferably, the signalsequence is from a mammal, such as from a native PRO polypeptide.

[0072] In a still further embodiment, the invention supplies an ex vivoproducer cell comprising a nucleic acid construct that expressesretroviral structural proteins and also comprises a retroviral vectorconsisting essentially of a promoter, nucleic acid encoding (a) a PROpolypeptide, (b) an agonist polypeptide of a PRO polypeptide or (c) anantagonist polypeptide of a PRO polypeptide, and a signal sequence forcellular secretion of the polypeptide, wherein said producer cellpackages the retroviral vector in association with the structuralproteins to produce recombinant retroviral particles.

[0073] In yet another embodiment, the invention provides a method forinhibiting endothelial cell growth in a mammal comprising administeringto the mammal (a) a PRO polypeptide, (b) an agonist of a PROpolypeptide, or (c) an antagonist of a PRO polypeptide, whereinendothelial cell growth in said mammal is inhibited, and wherein saidagonist or antagonist may be an anti-PRO antibody. Preferably, themammal is human and the endothelial cell growth is associated with atumor or a retinal disorder.

[0074] In yet another embodiment, the invention provides a method forstimulating endothelial cell growth in a mammal comprising administeringto the mammal (a) a PRO polypeptide, (b) an agonist of a PROpolypeptide, or (c) an antagonist of a PRO polypeptide, whereinendothelial cell growth in said mammal is stimulated, and wherein saidagonist or antagonist may be an anti-PRO antibody. Preferably, themammal is human.

[0075] In yet another embodiment, the invention provides a method forinhibiting cardiac hypertrophy in a mammal comprising administering tothe mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide,or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophyin said mammal is inhibited, and wherein said agonist or antagonist maybe an anti-PRO antibody. Preferably, the mammal is human and the cardiachypertrophy has been induced by myocardial infarction.

[0076] In yet another embodiment, the invention provides a method forstimulating cardiac hypertrophy in a mammal comprising administering tothe mammal (a) a PRO polypeptide, (b) an agonist of a PRO polypeptide,or (c) an antagonist of a PRO polypeptide, wherein cardiac hypertrophyin said mammal is stimulated, and wherein said agonist or antagonist maybe an anti-PRO antibody. Preferably, the mammal is human who suffersfrom congestive heart failure.

[0077] In yet another embodiment, the invention provides a method forinhibiting angiogenesis induced by a PRO polypeptide in a mammalcomprising administering a therapeutically effective amount of ananti-PRO antibody to the mammal. Preferably, the mammal is a human, andmore preferably the mammal has a tumor or a retinal disorder.

[0078] In yet another embodiment, the invention provides a method forstimulating angiogenesis induced by a PRO polypeptide in a mammalcomprising administering a therapeutically effective amount of a PROpolypeptide to the mammal. Preferably, the mammal is a human, and morepreferably angiogenesis would promote tissue regeneration or woundhealing.

[0079] In yet another embodiment, the invention provides a method formodulating (e.g., inhibiting or stimulating) endothelial cell growth ina mammal comprising administering to the mammal a PRO21, PRO181, PRO205,PRO214, PRO221, PRO229, PRO231, PRO238, PRO241, PRO247, PRO256, PRO258,PRO263, PRO265, PRO295, PRO321, PRO322, PRO337, PRO363, PRO365, PRO444,PRO533, PRO697, PRO720, PRO725, PRO771, PRO788, PRO791, PRO819, PRO827,PRO828, PRO836, PRO846, PRO865, PRO1005, PRO1006, PRO1007, PRO1025,PRO1029, PRO1054, PRO1071, PRO1075, PRO1079, PRO1080, PRO1114, PRO1131,PRO1155, PRO1160, PRO1184, PRO1186, PRO1190, PRO1192, PRO1195, PRO1244,PRO1272, PRO1273, PRO1274, PRO1279, PRO1283, PRO1286, PRO1306, PRO1309,PRO1325, PRO1329, PRO1347, PRO1356, PRO1376, PRO1382, PRO1411, PRO1412,PRO1419, PRO1474, PRO1477, PRO1488, PRO1508, PRO1550, PRO1556, PRO1760,PRO1782, PRO1787, PRO1801, PRO1868, PRO1887, PRO1890, PRO3438, PRO3444,PRO4302, PRO4324, PRO4333, PRO4341, PRO4342, PRO4353, PRO4354, PRO4356,PRO4371, PRO4405, PRO4408, PRO4422, PRO4425, PRO4499, PRO5723, PRO5725,PRO5737, PRO5776, PRO6006, PRO6029, PRO6071, PRO7436, PRO9771, PRO9821,PRO9873, PRO10008, PRO10096, PRO19670, PRO20040, PRO20044, PRO21055,PRO21384 or PRO28631 polypeptide, agonist or antagonist thereof, whereinendothelial cell growth in said mammal is modulated.

[0080] In yet another embodiment, the invention provides a method formodulating (e.g., inhibiting or stimulating) smooth muscle cell growthin a mammal comprising administering to the mammal a PRO162, PRO181,PRO182, PRO195, PRO204, PRO221, PRO230, PRO256, PRO258, PRO533, PRO697,PRO725, PRO738, PRO826, PRO836, PRO840, PRO846, PRO865, PRO982, PRO1025,PRO1029, PRO1071, PRO1080, PRO1083, PRO1134, PRO1160, PRO1182, PRO1184,PRO1186, PRO1192, PRO1265, PRO1274, PRO1279, PRO1283, PRO1306, PRO1308,PRO1309, PRO1325, PRO1337, PRO1338, PRO1343, PRO1376, PRO1387, PRO1411,PRO1412, PRO1415, PRO1434, PRO1474, PRO1488, PRO1550, PRO1556, PRO1567,PRO1600, PRO1754, PRO1758, PRO1760, PRO1787, PRO1865, PRO1868, PRO1917,PRO1928, PRO3438, PRO3562, PRO4302, PRO4333, PRO4345, PRO4353, PRO4354,PRO4405, PRO4408, PRO4430, PRO4503, PRO5725, PRO6714, PRO9771, PRO9820,PRO9940, PRO10096, PRO21055, PRO21184 or PRO21366 polypeptide, agonistor antagonist thereof, wherein endothelial cell growth in said mammal ismodulated.

[0081] In yet another embodiment, the invention provides a method formodulating (e.g., inducing or reducing) cardiac hypertrophy in a mammalcomprising administering to the mammal a PRO21 polypeptide, agonist orantagonist thereof, wherein cardiac hypertrophy in said mammal ismodulated.

[0082] In yet another embodiment, the invention provides a method formodulating (e.g., inducing or reducing) endothelial cell apoptosis in amammal comprising administering to the mammal a PRO4302 polypeptide,agonist or antagonist thereof, wherein cardiac hypertrophy in saidmammal is modulated.

[0083] In yet another embodiment, the invention provides a method formodulating (e.g., stimulating or inhibiting) angiogenesis in a mammalcomprising administering a therapeutically effective amount of a PRO1376or PRO1449 polypeptide, agonist or antagonist thereof to the mammal,wherein said angiogenesis is modulated.

[0084] In yet another embodiment, the invention provides a method formodulating (e.g., inducing or reducing) angiogenesis by modulating(e.g., inducing or reducing) endothelial cell tube formation in a mammalcomprising administering to the mammal a PRO178, PRO195, PRO228, PRO301,PRO302, PRO532, PRO724, PRO730, PRO734, PRO793, PRO871, PRO938, PRO1012,PRO1120, PRO1139, PRO1198, PRO1287, PRO1361, PRO1864, PRO1873, PRO2010,PRO3579, PRO4313, PRO4527, PRO4538, PRO4553, PRO4995, PRO5730, PRO6008,PRO7223, PRO7248 or PRO7261 polypeptide, agonist or antagonist thereof,wherein endothelial cell tube formation in said mammal is modulated.

[0085] In other embodiments of the present invention, the inventionprovides an isolated nucleic acid molecule comprising a nucleotidesequence that encodes a PRO polypeptide.

[0086] In one aspect, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%nucleic acid sequence identity and alternatively at least about 99%nucleic acid sequence identity to (a) a DNA molecule encoding a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein, or (b) the complement of the DNA molecule of (a).

[0087] In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%nucleic acid sequence identity and alternatively at least about 99%nucleic acid sequence identity to (a) a DNA molecule comprising thecoding sequence of a full-length PRO polypeptide cDNA as disclosedherein, the coding sequence of a PRO polypeptide lacking the signalpeptide as disclosed herein, the coding sequence of an extracellulardomain of a transmembrane PRO polypeptide, with or without the signalpeptide, as disclosed herein or the coding sequence of any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein, or (b) the complement of the DNA molecule of (a).

[0088] In a further aspect, the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence having at least about80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% or 98% nucleic acid sequence identity andalternatively at least about 99% nucleic acid sequence identity to (a) aDNA molecule that encodes the same mature polypeptide encoded by any ofthe human protein cDNAs deposited with the ATCC as disclosed herein, or(b) the complement of the DNA molecule of (a).

[0089] Another aspect of the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence encoding a PROpolypeptide which is either transmembrane domain-deleted ortransmembrane domain-inactivated, or is complementary to such encodingnucleotide sequence, wherein the transmembrane domain(s) of suchpolypeptide are disclosed herein. Therefore, soluble extracellulardomains of the herein described PRO polypeptides are contemplated.

[0090] Another embodiment is directed to fragments of a PRO polypeptidecoding sequence, or the complement thereof, that may find use as, forexample, hybridization probes, for encoding fragments of a PROpolypeptide that may optionally encode a polypeptide comprising abinding site for an anti-PRO antibody or as antisense oligonucleotideprobes. Such nucleic acid fragments are usually at least about 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 250, 300, 350, 400, 450, 500, 600, 700 or 800 nucleotides inlength and alternatively at least about 1000 nucleotides in length,wherein in this context the term “about” means the referenced nucleotidesequence length plus or minus 10% of that referenced length. It is notedthat novel fragments of a PRO polypeptide-encoding nucleotide sequencemay be determined in a routine manner by aligning the PROpolypeptide-encoding nucleotide sequence with other known nucleotidesequences using any of a number of well known sequence alignmentprograms and determining which PRO polypeptide-encoding nucleotidesequence fragment(s) are novel. All of such PRO polypeptide-encodingnucleotide sequences are contemplated herein. Also contemplated are thePRO polypeptide fragments encoded by these nucleotide moleculefragments, preferably those PRO polypeptide fragments that comprise abinding site for an anti-PRO antibody.

[0091] In another embodiment, the invention provides an isolated PROpolypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0092] In a certain aspect, the invention provides an isolated PROpolypeptide comprising an amino acid sequence having at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% or 98% amino acid sequence identity and alternatively atleast about 99% amino acid sequence identity to a PRO polypeptide havinga fill-length amino acid sequence as disclosed herein, an amino acidsequence lacking the signal peptide as disclosed herein, anextracellular domain of a transmembrane protein, with or without thesignal peptide, as disclosed herein or any other specifically definedfragment of the full-length amino acid sequence as disclosed herein.

[0093] In a further aspect, the invention provides an isolated PROpolypeptide comprising an amino acid sequence having at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%95%, 96%, 97% or 98% amino acid sequence identity and alternatively atleast about 99% amino acid sequence identity to an amino acid sequenceencoded by any of the human protein cDNAs deposited with the ATCC asdisclosed herein.

[0094] In a specific aspect, the invention provides an isolated PROpolypeptide without the N-terminal signal sequence and/or the initiatingmethionine and that is encoded by a nucleotide sequence that encodessuch an amino acid sequence as hereinbefore described. Processes forproducing the same are also herein described, wherein those processescomprise culturing a host cell comprising a vector which comprises theappropriate encoding nucleic acid molecule under conditions suitable forexpression of the PRO polypeptide and recovering the PRO polypeptidefrom the cell culture.

[0095] Another aspect of the invention provides an isolated PROpolypeptide which is either transmembrane domain-deleted ortransmembrane domain-inactivated. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO polypeptide and recovering the PRO polypeptide from the cellculture.

[0096] In yet another embodiment, the invention provides agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

[0097] In a further embodiment, the invention provides a method ofidentifying agonists or antagonists to a PRO polypeptide which comprisecontacting the PRO polypeptide with a candidate molecule and monitoringa biological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

[0098] In a still further embodiment, the invention provides acomposition of matter comprising a PRO polypeptide, or an agonist orantagonist of a PRO polypeptide as herein described, or an anti-PROantibody, in combination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

[0099] Another embodiment of the present invention is directed to theuse of a PRO polypeptide, or an agonist or antagonist thereof ashereinbefore described, or an anti-PRO antibody, for the preparation ofa medicament useful in the treatment of a condition which is responsiveto the PRO polypeptide, an agonist or antagonist thereof or an anti-PROantibody.

[0100] In additional embodiments of the present invention, the inventionprovides vectors comprising DNA encoding any of the herein describedpolypeptides. Host cells comprising any such vector are also provided.By way of example, the host cells may be CHO cells, E. Coli, yeast, orBaculovirus-infected insect cells. A process for producing any of theherein described polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of thedesired polypeptide and recovering the desired polypeptide from the cellculture.

[0101] In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

[0102] In yet another embodiment, the invention provides an antibodywhich specifically binds to any of the above or below describedpolypeptides. Optionally, the antibody is a monoclonal antibody,humanized antibody, antibody fragment or single-chain antibody.

[0103] In yet other embodiments, the invention provides oligonucleotideprobes useful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0104]FIG. 1 shows a nucleotide sequence (SEQ ID NO: 1) of a nativesequence PRO181 cDNA, wherein SEQ ID NO: 1 is a clone designated hereinas “DNA23330-1390”.

[0105]FIG. 2 shows the amino acid sequence (SEQ ID NO: 2) derived fromthe coding sequence of SEQ ID NO: 1 shown in FIG. 1.

[0106]FIG. 3 shows a nucleotide sequence (SEQ ID NO: 3) of a nativesequence PRO178 cDNA, wherein SEQ ID NO: 3 is a clone designated hereinas “DNA23339-1130”.

[0107]FIG. 4 shows the amino acid sequence (SEQ ID NO: 4) derived fromthe coding sequence of SEQ ID NO: 3 shown in FIG. 3.

[0108]FIG. 5 shows a nucleotide sequence (SEQ ID NO: 5) of a nativesequence PRO444 cDNA, wherein SEQ ID NO: 5 is a clone designated hereinas “DNA26846-1397”.

[0109]FIG. 6 shows the amino acid sequence (SEQ ID NO: 6) derived fromthe coding sequence of SEQ ID NO: 5 shown in FIG. 5.

[0110]FIG. 7 shows a nucleotide sequence (SEQ ID NO: 7) of a nativesequence PRO195 cDNA, wherein SEQ ID NO: 7 is a clone designated hereinas “DNA26847-1395”.

[0111]FIG. 8 shows the amino acid sequence (SEQ ID NO: 8) derived fromthe coding sequence of SEQ ID NO: 7 shown in FIG. 7.

[0112]FIG. 9 shows a nucleotide sequence (SEQ ID NO: 9) of a nativesequence PRO182 cDNA, wherein SEQ ID NO: 9 is a clone designated hereinas “DNA27865-1091”.

[0113]FIG. 10 shows the amino acid sequence (SEQ ID NO: 10) derived fromthe coding sequence of SEQ ID NO: 9 shown in FIG. 9.

[0114]FIG. 11 shows a nucleotide sequence (SEQ ID NO: 11) of a nativesequence PRO205 cDNA, wherein SEQ ID NO: 11 is a clone designated hereinas “DNA30868-1156”.

[0115]FIG. 12 shows the amino acid sequence (SEQ ID NO: 12) derived fromthe coding sequence of SEQ ID NO: 11 shown in FIG. 11.

[0116]FIG. 13 shows a nucleotide sequence (SEQ ID NO: 13) of a nativesequence PRO204 cDNA, wherein SEQ ID NO: 13 is a clone designated hereinas “DNA30871-1157”.

[0117]FIG. 14 shows the amino acid sequence (SEQ ID NO: 14) derived fromthe coding sequence of SEQ ID NO: 13 shown in FIG. 13.

[0118]FIG. 15 shows a nucleotide sequence (SEQ ID NO: 15) of a nativesequence PRO1873 cDNA, wherein SEQ ID NO: 15 is a clone designatedherein as “DNA30880”.

[0119]FIG. 16 shows the amino acid sequence (SEQ ID NO: 16) derived fromthe coding sequence of SEQ ID NO: 15 shown in FIG. 15.

[0120]FIG. 17 shows a nucleotide sequence (SEQ ID NO: 17) of a nativesequence PRO214 cDNA, wherein SEQ ID NO: 17 is a clone designated hereinas “DNA32286-1191 ”.

[0121]FIG. 18 shows the amino acid sequence (SEQ ID NO: 18) derived fromthe coding sequence of SEQ ID NO: 17 shown in FIG. 17.

[0122]FIG. 19 shows a nucleotide sequence (SEQ ID NO: 19) of a nativesequence PRO221 cDNA, wherein SEQ ID NO: 19 is a clone designated hereinas “DNA33089-1132”.

[0123]FIG. 20 shows the amino acid sequence (SEQ ID NO: 20) derived fromthe coding sequence of SEQ ID NO: 19 shown in FIG. 19.

[0124]FIG. 21 shows a nucleotide sequence (SEQ ID NO: 21) of a nativesequence PRO228 cDNA, wherein SEQ ID NO: 21 is a clone designated hereinas “DNA33092-1202”.

[0125]FIG. 22 shows the amino acid sequence (SEQ ID NO: 22) derived fromthe coding sequence of SEQ ID NO: 21 shown in FIG. 21.

[0126]FIG. 23 shows a nucleotide sequence (SEQ ID NO: 23) of a nativesequence PRO229 cDNA, wherein SEQ ID NO: 23 is a clone designated hereinas “DNA33100-1159”.

[0127]FIG. 24 shows the amino acid sequence (SEQ ID NO: 24) derived fromthe coding sequence of SEQ ID NO: 23 shown in FIG. 23.

[0128]FIG. 25 shows a nucleotide sequence (SEQ ID NO: 25) of a nativesequence PRO230 cDNA, wherein SEQ ID NO: 25 is a clone designated hereinas “DNA33223-1136”.

[0129]FIG. 26 shows the amino acid sequence (SEQ ID NO: 26) derived fromthe coding sequence of SEQ ID NO: 25 shown in FIG. 25.

[0130]FIG. 27 shows a nucleotide sequence (SEQ ID NO: 27) of a nativesequence PRO7223 cDNA, wherein SEQ ID NO: 27 is a clone designatedherein as “DNA34385”.

[0131]FIG. 28 shows the amino acid sequence (SEQ ID NO: 28) derived fromthe coding sequence of SEQ ID NO: 27 shown in FIG. 27.

[0132]FIG. 29 shows a nucleotide sequence (SEQ ID NO: 29) of a nativesequence PRO241 cDNA, wherein SEQ ID NO: 29 is a clone designated hereinas “DNA34392-1170”.

[0133]FIG. 30 shows the amino acid sequence (SEQ ID NO: 30) derived fromthe coding sequence of SEQ ID NO: 29 shown in FIG. 29.

[0134]FIG. 31 shows a nucleotide sequence (SEQ ID NO: 31) of a nativesequence PRO263 cDNA, wherein SEQ ID NO: 31 is a clone designated hereinas “DNA34431-1177”.

[0135]FIG. 32 shows the amino acid sequence (SEQ ID NO: 32) derived fromthe coding sequence of SEQ ID NO: 31 shown in FIG. 31.

[0136]FIG. 33 shows a nucleotide sequence (SEQ ID NO: 33) of a nativesequence PRO321 cDNA, wherein SEQ ID NO: 33 is a clone designated hereinas “DNA34433-1308”.

[0137]FIG. 34 shows the amino acid sequence (SEQ ID NO: 34) derived fromthe coding sequence of SEQ ID NO: 33 shown in FIG. 33.

[0138]FIG. 35 shows a nucleotide sequence (SEQ ID NO: 35) of a nativesequence PRO231 cDNA, wherein SEQ ID NO: 35 is a clone designated hereinas “DNA34434-1139”.

[0139]FIG. 36 shows the amino acid sequence (SEQ ID NO: 36) derived fromthe coding sequence of SEQ ID NO: 35 shown in FIG. 35.

[0140]FIG. 37 shows a nucleotide sequence (SEQ ID NO: 37) of a nativesequence PRO238 cDNA, wherein SEQ ID NO: 37 is a clone designated hereinas “DNA35600-1162”.

[0141]FIG. 38 shows the amino acid sequence (SEQ ID NO: 38) derived fromthe coding sequence of SEQ ID NO: 37 shown in FIG. 37.

[0142]FIG. 39 shows a nucleotide sequence (SEQ ID NO: 39) of a nativesequence PRO247 cDNA, wherein SEQ ID NO: 39 is a clone designated hereinas “DNA35673-1201”.

[0143]FIG. 40 shows the amino acid sequence (SEQ ID NO: 40) derived fromthe coding sequence of SEQ ID NO: 39 shown in FIG. 39.

[0144]FIG. 41 shows a nucleotide sequence (SEQ ID NO: 41) of a nativesequence PRO256 cDNA, wherein SEQ ID NO: 41 is a clone designated hereinas “DNA35880-1160”.

[0145]FIG. 42 shows the amino acid sequence (SEQ ID NO: 42) derived fromthe coding sequence of SEQ ID NO: 41 shown in FIG. 41.

[0146]FIG. 43 shows a nucleotide sequence (SEQ ID NO: 43) of a nativesequence PRO258 cDNA, wherein SEQ ID NO: 43 is a clone designated hereinas “DNA35918-1174”.

[0147]FIG. 44 shows the amino acid sequence (SEQ ID NO: 44) derived fromthe coding sequence of SEQ ID NO: 43 shown in FIG. 43.

[0148]FIG. 45 shows a nucleotide sequence (SEQ ID NO: 45) of a nativesequence PRO265 cDNA, wherein SEQ ID NO: 45 is a clone designated hereinas “DNA36350-1158”.

[0149]FIG. 46 shows the amino acid sequence (SEQ ID NO: 46) derived fromthe coding sequence of SEQ ID NO: 45 shown in FIG. 45.

[0150]FIG. 47 shows a nucleotide sequence (SEQ ID NO: 47) of a nativesequence PRO21 cDNA, wherein SEQ ID NO: 47 is a clone designated hereinas “DNA36638-1056”.

[0151]FIG. 48 shows the amino acid sequence (SEQ ID NO: 48) derived fromthe coding sequence of SEQ ID NO: 47 shown in FIG. 47.

[0152]FIG. 49 shows a nucleotide sequence (SEQ ID NO: 49) of a nativesequence PRO295 cDNA, wherein SEQ ID NO: 49 is a clone designated hereinas “DNA38268-1188”.

[0153]FIG. 50 shows the amino acid sequence (SEQ ID NO: 50) derived fromthe coding sequence of SEQ ID NO: 49 shown in FIG. 49.

[0154]FIG. 51 shows a nucleotide sequence (SEQ ID NO: 51) of a nativesequence PRO302 cDNA, wherein SEQ ID NO: 51 is a clone designated hereinas “DNA40370-1217”.

[0155]FIG. 52 shows the amino acid sequence (SEQ ID NO: 52) derived fromthe coding sequence of SEQ ID NO: 51 shown in FIG. 51.

[0156]FIG. 53 shows a nucleotide sequence (SEQ ID NO: 53) of a nativesequence PRO301 cDNA, wherein SEQ ID NO: 53 is a clone designated hereinas “DNA40628-1216”.

[0157]FIG. 54 shows the amino acid sequence (SEQ ID NO: 54) derived fromthe coding sequence of SEQ ID NO: 53 shown in FIG. 53.

[0158]FIG. 55 shows a nucleotide sequence (SEQ ID NO: 55) of a nativesequence PRO337 cDNA, wherein SEQ ID NO: 55 is a clone designated hereinas “DNA43316-1237”.

[0159]FIG. 56 shows the amino acid sequence (SEQ ID NO: 56) derived fromthe coding sequence of SEQ ID NO: 55 shown in FIG. 55.

[0160]FIG. 57 shows a nucleotide sequence (SEQ ID NO: 57) of a nativesequence PRO7248 cDNA, wherein SEQ ID NO: 57 is a clone designatedherein as “DNA44195”.

[0161]FIG. 58 shows the amino acid sequence (SEQ ID NO: 58) derived fromthe coding sequence of SEQ ID NO: 57 shown in FIG. 57.

[0162]FIG. 59 shows a nucleotide sequence (SEQ ID NO: 59) of a nativesequence PRO846 cDNA, wherein SEQ ID NO: 59 is a clone designated hereinas “DNA44196-1353”.

[0163]FIG. 60 shows the amino acid sequence (SEQ ID NO: 60) derived fromthe coding sequence of SEQ ID NO: 59 shown in FIG. 59.

[0164]FIG. 61 shows a nucleotide sequence (SEQ ID NO: 61) of a nativesequence PRO1864 cDNA, wherein SEQ ID NO: 61 is a clone designatedherein as “DNA45409-251”.

[0165]FIG. 62 shows the amino acid sequence (SEQ ID NO: 62) derived fromthe coding sequence of SEQ ID NO: 61 shown in FIG. 61.

[0166]FIG. 63 shows a nucleotide sequence (SEQ ID NO: 63) of a nativesequence PRO363 cDNA, wherein SEQ ID NO: 63 is a clone designated hereinas “DNA45419-1252”.

[0167]FIG. 64 shows the amino acid sequence (SEQ ID NO: 64) derived fromthe coding sequence of SEQ ID NO: 63 shown in FIG. 63.

[0168]FIG. 65 shows a nucleotide sequence (SEQ ID NO: 65) of a nativesequence PRO730 cDNA, wherein SEQ ID NO: 65 is a clone designated hereinas “DNA45624-1400”.

[0169]FIG. 66 shows the amino acid sequence (SEQ ID NO: 66) derived fromthe coding sequence of SEQ ID NO: 65 shown in FIG. 65.

[0170]FIG. 67 shows a nucleotide sequence (SEQ ID NO: 67) of a nativesequence PRO365 cDNA, wherein SEQ ID NO: 67 is a clone designated hereinas “DNA46777-1253”.

[0171]FIG. 68 shows the amino acid sequence (SEQ ID NO: 68) derived fromthe coding sequence of SEQ ID NO: 67 shown in FIG. 67.

[0172]FIG. 69 shows a nucleotide sequence (SEQ ID NO: 69) of a nativesequence PRO532 cDNA, wherein SEQ ID NO: 69 is a clone designated hereinas “DNA48335”.

[0173]FIG. 70 shows the amino acid sequence (SEQ ID NO: 70) derived fromthe coding sequence of SEQ ID NO: 69 shown in FIG. 69.

[0174]FIG. 71 shows a nucleotide sequence (SEQ ID NO: 71) of a nativesequence PRO322 cDNA, wherein SEQ ID NO: 71 is a clone designated hereinas “DNA48336-1309”.

[0175]FIG. 72 shows the amino acid sequence (SEQ ID NO: 72) derived fromthe coding sequence of SEQ ID NO: 71 shown in FIG. 71.

[0176]FIG. 73 shows a nucleotide sequence (SEQ ID NO: 73) of a nativesequence PRO1120 cDNA, wherein SEQ ID NO: 73 is a clone designatedherein as “DNA48606-1479”.

[0177]FIG. 74 shows the amino acid sequence (SEQ ID NO: 74) derived fromthe coding sequence of SEQ ID NO: 73 shown in FIG. 73.

[0178]FIG. 75 shows a nucleotide sequence (SEQ ID NO: 75) of a nativesequence PRO7261 cDNA, wherein SEQ ID NO: 75 is a clone designatedherein as “DNA49149”.

[0179]FIG. 76 shows the amino acid sequence (SEQ ID NO: 76) derived fromthe coding sequence of SEQ ID NO: 75 shown in FIG. 75.

[0180]FIG. 77 shows a nucleotide sequence (SEQ ID NO: 77) of a nativesequence PRO533 cDNA, wherein SEQ ID NO: 77 is a clone designated hereinas “DNA49435-1219”.

[0181]FIG. 78 shows the amino acid sequence (SEQ ID NO: 78) derived fromthe coding sequence of SEQ ID NO: 77 shown in FIG. 77.

[0182]FIG. 79 shows a nucleotide sequence (SEQ ID NO: 79) of a nativesequence PRO724 cDNA, wherein SEQ ID NO: 79 is a clone designated hereinas “DNA49631-1328”.

[0183]FIG. 80 shows the amino acid sequence (SEQ ID NO: 80) derived fromthe coding sequence of SEQ ID NO: 79 shown in FIG. 79.

[0184]FIG. 81 shows a nucleotide sequence (SEQ ID NO: 81) of a nativesequence PRO734 cDNA, wherein SEQ ID NO: 81 is a clone designated hereinas “DNA49817”.

[0185]FIG. 82 shows the amino acid sequence (SEQ ID NO: 82) derived fromthe coding sequence of SEQ ID NO: 81 shown in FIG. 81.

[0186]FIG. 83 shows a nucleotide sequence (SEQ ID NO: 83) of a nativesequence PRO771 cDNA, wherein SEQ ID NO: 83 is a clone designated hereinas “DNA49829-1346”.

[0187]FIG. 84 shows the amino acid sequence (SEQ ID NO: 84) derived fromthe coding sequence of SEQ ID NO: 83 shown in FIG. 83.

[0188]FIG. 85 shows a nucleotide sequence (SEQ ID NO: 85) of a nativesequence PRO2010 cDNA, wherein SEQ ID NO: 85 is a clone designatedherein as “DNA50792”.

[0189]FIG. 86 shows the amino acid sequence (SEQ ID NO: 86) derived fromthe coding sequence of SEQ ID NO: 85 shown in FIG. 85.

[0190]FIG. 87 shows a nucleotide sequence (SEQ ID NO: 87) of a nativesequence PRO871 cDNA, wherein SEQ ID NO: 87 is a clone designated hereinas “DNA50919-1361”.

[0191]FIG. 88 shows the amino acid sequence (SEQ ID NO: 88) derived fromthe coding sequence of SEQ ID NO: 87 shown in FIG. 87.

[0192]FIG. 89 shows a nucleotide sequence (SEQ ID NO: 89) of a nativesequence PRO697 cDNA, wherein SEQ ID NO: 89 is a clone designated hereinas “DNA50920-1325”.

[0193]FIG. 90 shows the amino acid sequence (SEQ ID NO: 90) derived fromthe coding sequence of SEQ ID NO: 89 shown in FIG. 89.

[0194]FIG. 91 shows a nucleotide sequence (SEQ ID NO: 91) of a nativesequence PRO1083 cDNA, wherein SEQ ID NO: 91 is a clone designatedherein as “DNA50921-1458”.

[0195]FIG. 92 shows the amino acid sequence (SEQ ID NO: 22) derived fromthe coding sequence of SEQ ID NO: 91 shown in FIG. 91.

[0196]FIG. 93 shows a nucleotide sequence (SEQ ID NO: 93) of a nativesequence PRO725 cDNA, wherein SEQ ID NO: 93 is a clone designated hereinas “DNA52758-1399”.

[0197]FIG. 94 shows the amino acid sequence (SEQ ID NO: 94) derived fromthe coding sequence of SEQ ID NO: 93 shown in FIG. 93.

[0198]FIG. 95 shows a nucleotide sequence (SEQ ID NO: 95) of a nativesequence PRO720 cDNA, wherein SEQ ID NO: 95 is a clone designated hereinas “DNA53517-1366-1”.

[0199]FIG. 96 shows the amino acid sequence (SEQ ID NO: 96) derived fromthe coding sequence of SEQ ID NO: 95 shown in FIG. 95.

[0200]FIG. 97 shows a nucleotide sequence (SEQ ID NO: 97) of a nativesequence PRO738 cDNA, wherein SEQ ID NO: 97 is a clone designated hereinas “DNA53915-1258”.

[0201]FIG. 98 shows the amino acid sequence (SEQ ID NO: 98) derived fromthe coding sequence of SEQ ID NO: 97 shown in FIG. 97.

[0202]FIG. 99 shows a nucleotide sequence (SEQ ID NO: 99) of a nativesequence PRO865 cDNA, wherein SEQ ID NO: 99 is a clone designated hereinas “DNA53974-1401”.

[0203]FIG. 100 shows the amino acid sequence (SEQ ID NO: 100) derivedfrom the coding sequence of SEQ ID NO: 99 shown in FIG. 99.

[0204]FIG. 101 shows a nucleotide sequence (SEQ ID NO: 101) of a nativesequence PRO840 cDNA, wherein SEQ ID NO: 101 is a clone designatedherein as “DNA53987-1438”.

[0205]FIG. 102 shows the amino acid sequence (SEQ ID NO: 102) derivedfrom the coding sequence of SEQ ID NO: 101 shown in FIG. 101.

[0206]FIG. 103 shows a nucleotide sequence (SEQ ID NO: 103) of a nativesequence PRO1080 cDNA, wherein SEQ ID NO: 103 is a clone designatedherein as “DNA56047-1456”.

[0207]FIG. 104 shows the amino acid sequence (SEQ ID NO: 104) derivedfrom the coding sequence of SEQ ID NO: 103 shown in FIG. 103.

[0208]FIG. 105 shows a nucleotide sequence (SEQ ID NO: 105) of a nativesequence PRO1079 cDNA, wherein SEQ ID NO: 105 is a clone designatedherein as “DNA56050-1455”.

[0209]FIG. 106 shows the amino acid sequence (SEQ ID NO: 106) derivedfrom the coding sequence of SEQ ID NO: 105 shown in FIG. 105.

[0210]FIG. 107 shows a nucleotide sequence (SEQ ID NO: 107) of a nativesequence PRO793 cDNA, wherein SEQ ID NO: 107 is a clone designatedherein as “DNA56110-1437”.

[0211]FIG. 108 shows the amino acid sequence (SEQ ID NO: 108) derivedfrom the coding sequence of SEQ ID NO: 107 shown in FIG. 107.

[0212]FIG. 109 shows a nucleotide sequence (SEQ ID NO: 109) of a nativesequence PRO788 cDNA, wherein SEQ ID NO: 109 is a clone designatedherein as “DNA56405-1357”.

[0213]FIG. 110 shows the amino acid sequence (SEQ ID NO: 110) derivedfrom the coding sequence of SEQ ID NO: 109 shown in FIG. 109.

[0214]FIG. 111 shows a nucleotide sequence (SEQ ID NO: 111) of a nativesequence PRO938 cDNA, wherein SEQ ID NO: 111 is a clone designatedherein as “DNA56433-1406”.

[0215]FIG. 112 shows the amino acid sequence (SEQ ID NO: 112) derivedfrom the coding sequence of SEQ ID NO: 111 shown in FIG. 111.

[0216]FIG. 113 shows a nucleotide sequence (SEQ ID NO: 113) of a nativesequence PRO1012 cDNA, wherein SEQ ID NO: 113 is a clone designatedherein as “DNA56439-1376”.

[0217]FIG. 114 shows the amino acid sequence (SEQ ID NO: 114) derivedfrom the coding sequence of SEQ ID NO: 113 shown in FIG. 113.

[0218]FIG. 115 shows a nucleotide sequence (SEQ ID NO: 115) of a nativesequence PRO1477 cDNA, wherein SEQ ID NO: 115 is a clone designatedherein as “DNA56529-1647”.

[0219]FIG. 116 shows the amino acid sequence (SEQ ID NO: 116) derivedfrom the coding sequence of SEQ ID NO: 115 shown in FIG. 115.

[0220]FIG. 117 shows a nucleotide sequence (SEQ ID NO: 117) of a nativesequence PRO1134 cDNA, wherein SEQ ID NO: 117 is a clone designatedherein as “DNA56865-1491”.

[0221]FIG. 118 shows the amino acid sequence (SEQ ID NO: 118) derivedfrom the coding sequence of SE ID NO: 117 shown in FIG. 117.

[0222]FIG. 119 shows a nucleotide sequence (SEQ ID NO: 119) of a nativesequence PRO162 cDNA, wherein SEQ ID NO: 119 is a clone designatedherein as “DNA56965-1356”.

[0223]FIG. 120 shows the amino acid sequence (SEQ ID NO: 120) derivedfrom the coding sequence of SEQ ID NO: 119 shown in FIG. 119.

[0224]FIG. 121 shows a nucleotide sequence (SEQ ID NO: 121) of a nativesequence PRO1114 cDNA, wherein SEQ ID NO: 121 is a clone designatedherein as “DNA57033-1403-1”.

[0225]FIG. 122 shows the amino acid sequence (SEQ ID NO: 122) derivedfrom the coding sequence of SEQ ID NO: 121 shown in FIG. 121.

[0226]FIG. 123 shows a nucleotide sequence (SEQ ID NO: 123) of a nativesequence PRO828 cDNA, wherein SEQ ID NO: 123 is a clone designatedherein as “DNA57037-1444”.

[0227]FIG. 124 shows the amino acid sequence (SEQ ID NO: 124) derivedfrom the coding sequence of SEQ ID NO: 123 shown in FIG. 123.

[0228]FIG. 125 shows a nucleotide sequence (SEQ ID NO: 125) of a nativesequence PRO827 cDNA, wherein SEQ ID NO: 125 is a clone designatedherein as “DNA57039-1402”.

[0229]FIG. 126 shows the amino acid sequence (SEQ ID NO: 126) derivedfrom the coding sequence of SEQ ID NO: 125 shown in FIG. 125.

[0230]FIG. 127 shows an nucleotide sequence (SEQ ID NO: 127) of a nativesequence PRO1075 cDNA, wherein SEQ ID NO: 127 is a clone designatedherein as “DNA57689-1385”.

[0231]FIG. 128 shows the amino acid sequence (SEQ ID NO: 128) derivedfrom the coding sequence of SEQ ID NO: 127 shown in FIG. 127.

[0232]FIG. 129 shows a nucleotide sequence (SEQ ID NO: 129) of a nativesequence PRO1007 cDNA, wherein SEQ ID NO: 129 is a clone designatedherein as “DNA57690-1374”.

[0233]FIG. 130 shows the amino acid sequence (SEQ ID NO: 130) derivedfrom the coding sequence of SEQ ID NO: 129 shown in FIG. 129.

[0234]FIG. 131 shows a nucleotide sequence (SEQ ID NO: 131) of a nativesequence PRO826 cDNA, wherein SEQ ID NO: 131 is a clone designatedherein as “DNA57694-1341”.

[0235]FIG. 132 shows the amino acid sequence (SEQ ID NO: 132) derivedfrom the coding sequence of SEQ ID NO: 131 shown in FIG. 131.

[0236]FIG. 133 shows a nucleotide sequence (SEQ ID NO: 133) of a nativesequence PRO819 cDNA, wherein SEQ ID NO: 132 is a clone designatedherein as “DNA57695-1340”.

[0237]FIG. 134 shows the amino acid sequence (SEQ ID NO: 134) derivedfrom the coding sequence of SEQ ID NO: 133 shown in FIG. 133.

[0238]FIG. 135 shows a nucleotide sequence (SEQ ID NO: 135) of a nativesequence PRO1006 cDNA, wherein SEQ ID NO: 135 is a clone designatedherein as “DNA57699-1412”.

[0239]FIG. 136 shows the amino acid sequence (SEQ ID NO: 136) derivedfrom the coding sequence of SEQ ID NO: 135 shown in FIG. 135.

[0240]FIG. 137 shows a nucleotide sequence (SEQ ID NO: 137) of a nativesequence PRO982 cDNA,wherein SEQ ID NO: 137 is a clone designated hereinas “DNA57700-1408”.

[0241]FIG. 138 shows the amino acid sequence (SEQ ID NO: 138) derivedfrom the coding sequence of SEQ ID NO: 137 shown in FIG. 137.

[0242]FIG. 139 shows a nucleotide sequence (SEQ ID NO: 139) of a nativesequence PRO1005 cDNA, wherein SEQ ID NO: 139 is a clone designatedherein as “DNA57708-1411”.

[0243]FIG. 140 shows the amino acid sequence (SEQ ID NO: 140) derivedfrom the coding sequence of SEQ ID NO: 139 shown in FIG. 139.

[0244]FIG. 141 shows a nucleotide sequence (SEQ ID NO: 141) of a nativesequence PRO791 cDNA, wherein SEQ ID NO: 141 is a clone designatedherein as “DNA57838-1337”.

[0245]FIG. 142 shows the amino acid sequence (SEQ ID NO: 142) derivedfrom the coding sequence of SEQ ID NO: 141 shown in FIG. 141.

[0246]FIG. 143 shows a nucleotide sequence (SEQ ID NO: 143) of a nativesequence PRO1071 cDNA, wherein SEQ ID NO: 143 is a clone designatedherein as “DNA58847-1383”.

[0247]FIG. 144 shows the amino acid sequence (SEQ ID NO: 144) derivedfrom the coding sequence of SEQ ID NO: 143 shown in FIG. 43.

[0248]FIG. 145 shows a nucleotide sequence (SEQ ID NO: 145) of a nativesequence PRO1415 cDNA, wherein SEQ ID NO: 145 is a clone designatedherein as “DNA58852-1637”.

[0249]FIG. 146 shows the amino acid sequence (SEQ ID NO: 146) derivedfrom the coding sequence of SEQ ID NO: 145 shown in FIG. 145.

[0250]FIG. 147 shows a nucleotide sequence (SEQ ID NO: 147) of a nativesequence PRO1054 cDNA, wherein SEQ ID NO: 147 is a clone designatedherein as “DNA58853-1423”.

[0251]FIG. 148 shows the amino acid sequence (SEQ ID NO: 148) derivedfrom the coding sequence of SEQ ID NO: 147 shown in FIG. 147.

[0252]FIG. 149 shows a nucleotide sequence (SEQ ID NO: 149) of a nativesequence PRO1411 cDNA, wherein SEQ ID NO: 149 is a clone designatedherein as “DNA59212-1627”.

[0253]FIG. 150 shows the amino acid sequence (SEQ ID NO: 150) derivedfrom the coding sequence of SEQ ID NO: 149 shown in FIG. 149.

[0254]FIG. 151 shows a nucleotide sequence (SEQ ID NO: 151) of a nativesequence PRO1184 cDNA, wherein SEQ ID NO: 151 is a clone designatedherein as “DNA59220-1514”.

[0255]FIG. 152 shows the amino acid sequence (SEQ ID NO: 152) derivedfrom the coding sequence of SEQ ID NO: 151 shown in FIG. 151.

[0256]FIG. 153 shows the nucleotide sequence (SEQ ID NO: 153) of anative sequence PRO1029 cDNA, wherein SEQ ID NO: 153 is a clonedesignated herein as “DNA59493-1420”.

[0257]FIG. 154 shows the amino acid sequence (SEQ ID NO: 154) derivedfrom the coding sequence of SEQ ID NO: 153 shown in FIG. 153.

[0258]FIG. 155 shows the nucleotide sequence (SEQ ID NO: 155) of anative sequence PRO1139 cDNA, wherein SEQ ID NO: 155 is a clonedesignated herein as “DNA59497-1496”.

[0259]FIG. 156 shows the amino acid sequence (SEQ ID NO: 156) derivedfrom the coding sequence of SEQ ID NO: 155 shown in FIG. 155.

[0260]FIG. 157 shows a nucleotide sequence (SEQ ID NO: 157) of a nativesequence PRO1190 cDNA, wherein SEQ ID NO: 157 is a clone designatedherein as “DNA59586-1520”.

[0261]FIG. 158 shows the amino acid sequence (SEQ ID NO: 158) derivedfrom the coding sequence of SEQ ID NO: 157 shown in FIG. 157.

[0262]FIG. 159 shows a nucleotide sequence (SEQ ID NO: 159) of a nativesequence PRO1309 cDNA, wherein SEQ ID NO: 159 is a clone designatedherein as “DNA59588-1571”.

[0263]FIG. 160 shows the amino acid sequence (SEQ ID NO: 160) derivedfrom the coding sequence of SEQ ID NO: 159 shown in FIG. 159.

[0264]FIG. 161 shows a nucleotide sequence (SEQ ID NO: 161) of a nativesequence PRO836 cDNA, wherein SEQ ID NO: 161 is a clone designatedherein as “DNA59620-1463”.

[0265]FIG. 162 shows the amino acid sequence (SEQ ID NO: 162) derivedfrom the coding sequence of SEQ ID NO: 161 shown in FIG. 161.

[0266]FIG. 163 shows a nucleotide sequence (SEQ ID NO: 163) of a nativesequence PRO) 1025 cDNA, wherein SEQ ID NO: 163 is a clone designatedherein as “DNA59622-1334”.

[0267]FIG. 164 shows the amino acid sequence (SEQ ID NO: 164) derivedfrom the coding sequence of SEQ ID NO: 163 shown in FIG. 163.

[0268]FIG. 165 shows a nucleotide sequence (SEQ ID NO: 165) of a nativesequence PRO) 1131 cDNA, wherein SEQ ID NO: 165 is a clone designatedherein as “DNA59777-1480”.

[0269]FIG. 166 shows the amino acid sequence (SEQ ID NO: 166) derivedfrom the coding sequence of SEQ ID NO: 165 shown in FIG. 165.

[0270]FIG. 167 shows a nucleotide sequence (SEQ ID NO: 167) of a nativesequence PRO) 1182 cDNA, wherein SEQ ID NO: 167 is a clone designatedherein as “DNA59848-1512”.

[0271]FIG. 168 shows the amino acid sequence (SEQ ID NO: 168) derivedfrom the coding sequence of SEQ ID NO: 167 shown in FIG. 167.

[0272]FIG. 169 shows a nucleotide sequence (SEQ ID NO: 169) of a nativesequence PRO1155 cDNA, wherein SEQ ID NO: 169 is a clone designatedherein as “DNA59849-1504”.

[0273]FIG. 170 shows the amino acid sequence (SEQ ID NO: 170) derivedfrom the coding sequence of SEQ ID NO: 169 shown in FIG. 169.

[0274]FIG. 171 shows a nucleotide sequence (SEQ ID NO: 171) of a nativesequence PRO1186 cDNA, wherein SEQ ID NO: 171 is a clone designatedherein as “DNA60621-1516”.

[0275]FIG. 172 shows the amino acid sequence (SEQ ID NO: 172) derivedfrom the coding sequence of SEQ ID NO: 171 shown in FIG. 171.

[0276]FIG. 173 shows a nucleotide sequence (SEQ ID NO: 173) of a nativesequence PRO1198 cDNA, wherein SEQ ID NO: 173 is a clone designatedherein as “DNA60622-1525”.

[0277]FIG. 174 shows the amino acid sequence (SEQ ID NO: 174) derivedfrom the coding sequence of SEQ ID NO: 173 shown in FIG. 173.

[0278]FIG. 175 shows a nucleotide sequence (SEQ ID NO: 175) of a nativesequence PRO1265 cDNA, wherein SEQ ID NO: 175 is a clone designatedherein as “DNA60764-1533”.

[0279]FIG. 176 shows the amino acid sequence (SEQ ID NO: 176) derivedfrom the coding sequence of SEQ ID NO: 175 shown in FIG. 175.

[0280]FIG. 177 shows a nucleotide sequence (SEQ ID NO: 177) of a nativesequence PRO1361 cDNA, wherein SEQ ID NO: 177 is a clone designatedherein as “DNA60783-1611”.

[0281]FIG. 178 shows the amino acid sequence (SEQ ID NO: 176) derivedfrom the coding sequence of SEQ ID NO: 177 shown in FIG. 177.

[0282]FIG. 179 shows a nucleotide sequence (SEQ ID NO: 179) of a nativesequence PRO1287 cDNA, wherein SEQ ID NO: 179 is a clone designatedherein as “DNA61755-1554”.

[0283]FIG. 180 shows the amino acid sequence (SEQ ID NO: 180) derivedfrom the coding sequence of SEQ ID NO: 179 shown in FIG. 179.

[0284]FIG. 181 shows a nucleotide sequence (SEQ ID NO: 181) of a nativesequence PRO1308 cDNA, wherein SEQ ID NO: 181 is a clone designatedherein as “DNA62306-1570”.

[0285]FIG. 182 shows the amino acid sequence (SEQ ID NO: 182) derivedfrom the coding sequence of SEQ ID NO: 181 shown in FIG. 181.

[0286]FIG. 183 shows a nucleotide sequence (SEQ ID NO: 183) of a nativesequence PRO4313 cDNA, wherein SEQ ID NO: 183 is a clone designatedherein as “DNA62312-2558”.

[0287]FIG. 184 shows the amino acid sequence (SEQ ID NO: 184) derivedfrom the coding sequence of SEQ ID NO: 183 shown in FIG. 183.

[0288]FIG. 185 shows a nucleotide sequence (SEQ ID NO: 185) of a nativesequence PRO1192 cDNA, wherein SEQ ID NO: 185 is a clone designatedherein as “DNA62814-1521 ”.

[0289]FIG. 186 shows the amino acid sequence (SEQ ID NO: 186) derivedfrom the coding sequence of SEQ ID NO: 185 shown in FIG. 185.

[0290]FIG. 187 shows a nucleotide sequence (SEQ ID NO: 187) of a nativesequence PRO1160 cDNA, wherein SEQ ID NO: 187 is a clone designatedherein as “DNA62872-1509”.

[0291]FIG. 188 shows the amino acid sequence (SEQ ID NO: 188) derivedfrom the coding sequence of SEQ ID NO: 187 shown in FIG. 187.

[0292]FIG. 189 shows a nucleotide sequence (SEQ ID NO: 189) of a nativesequence PRO1244 cDNA, wherein SEQ ID NO: 189 is a clone designatedherein as “DNA64883-1526”.

[0293]FIG. 190 shows the amino acid sequence (SEQ ID NO: 190) derivedfrom the coding sequence of SEQ ID NO: 189 shown in FIG. 189.

[0294]FIG. 191 shows a nucleotide sequence (SEQ ID NO: 191) of a nativesequence PRO1356 cDNA, wherein SEQ ID NO: 191 is a clone designatedherein as “DNA64886-1601”.

[0295]FIG. 192 shows the amino acid sequence (SEQ ID NO: 192) derivedfrom the coding sequence of SEQ ID NO: 191 shown in FIG. 191.

[0296]FIG. 193 shows a nucleotide sequence (SEQ ID NO: 193) of a nativesequence PRO1274 cDNA, wherein SEQ ID NO: 193 is a clone designatedherein as “DNA64889-1541”.

[0297]FIG. 194 shows the amino acid sequence (SEQ ID NO: 194) derivedfrom the coding sequence of SEQ ID NO: 193 shown in FIG. 193.

[0298]FIG. 195 shows a nucleotide sequence (SEQ ID NO: 195) of a nativesequence PRO1272 cDNA, wherein SEQ ID NO: 195 is a clone designatedherein as “DNA64896-1539”.

[0299]FIG. 196 shows the amino acid sequence (SEQ ID NO: 196) derivedfrom the coding sequence of SEQ ID NO: 195 shown in FIG. 195.

[0300]FIG. 197 shows a nucleotide sequence (SEQ ID NO: 197) of a nativesequence PRO1412 cDNA, wherein SEQ ID NO: 197 is a clone designatedherein as “DNA64897-1628 ”.

[0301]FIG. 198 shows the amino acid sequence (SEQ ID NO: 198) derivedfrom the coding sequence of SEQ ID NO: 197 shown in FIG. 197.

[0302]FIG. 199 shows a nucleotide sequence (SEQ ID NO: 199) of a nativesequence PRO1286 cDNA, wherein SEQ ID NO: 199 is a clone designatedherein as “DNA64903-1553”.

[0303]FIG. 200 shows the amino acid sequence (SEQ ID NO: 200) derivedfrom the coding sequence of SEQ ID NO: 199 shown in FIG. 199.

[0304]FIG. 201 shows a nucleotide sequence (SEQ ID NO: 201) of a nativesequence PRO1347 cDNA, wherein SEQ ID NO: 201 is a clone designatedherein as “DNA64950-1590”.

[0305]FIG. 202 shows the amino acid sequence (SEQ ID NO: 202) derivedfrom the coding sequence of SEQ ID NO: 201 shown in FIG. 201.

[0306]FIG. 203 shows a nucleotide sequence (SEQ ID NO: 203) of a nativesequence PRO1273 cDNA, wherein SEQ ID NO: 203 is a clone designatedherein as “DNA65402-1540”.

[0307]FIG. 204 shows the amino acid sequence (SEQ ID NO: 204) derivedfrom the coding sequence of SEQ ID NO: 203 shown in FIG. 203.

[0308]FIG. 205 shows a nucleotide sequence (SEQ ID NO: 205) of a nativesequence PRO1283 cDNA, wherein SEQ ID NO: 205 is a clone designatedherein as “DNA65404-1551”.

[0309]FIG. 206 shows the amino acid sequence (SEQ ID NO: 206) derivedfrom the coding sequence of SEQ ID NO: 205 shown in FIG. 205.

[0310]FIG. 207 shows a nucleotide sequence (SEQ ID NO: 207) of a nativesequence PRO1279 cDNA, wherein SEQ ID NO: 207 is a clone designatedherein as “DNA65405-1547”.

[0311]FIG. 208 shows the amino acid sequence (SEQ ID NO: 208) derivedfrom the coding sequence of SEQ ID NO: 207 shown in FIG. 207.

[0312]FIG. 209 shows a nucleotide sequence (SEQ ID NO: 209) of a nativesequence PRO1306 cDNA, wherein SEQ ID NO: 209 is a clone designatedherein as “DNA65410-1569”.

[0313]FIG. 210 shows the amino acid sequence (SEQ ID NO: 210) derivedfrom the coding sequence of SEQ ID NO: 209 shown in FIG. 209.

[0314]FIG. 211 shows a nucleotide sequence (SEQ ID NO: 211) of a nativesequence PRO1195 cDNA, wherein SEQ ID NO: 211 is a clone designatedherein as “DNA65412-1523”.

[0315]FIG. 212 shows the amino acid sequence (SEQ ID NO: 212) derivedfrom the coding sequence of SEQ ID NO: 211 shown in FIG. 211.

[0316]FIG. 213 shows a nucleotide sequence (SEQ ID NO: 213) of a nativesequence PRO4995 cDNA, wherein SEQ ID NO: 213 is a clone designatedherein as “DNA66307-2661”.

[0317]FIG. 214 shows the amino acid sequence (SEQ ID NO: 214) derivedfrom the coding sequence of SEQ ID NO: 213 shown in FIG. 213.

[0318]FIG. 215 shows a nucleotide sequence (SEQ ID NO: 215) of a nativesequence PRO1382 cDNA, wherein SEQ ID NO: 215 is a clone designatedherein as “DNA66526-1616”.

[0319]FIG. 216 shows the amino acid sequence (SEQ ID NO: 216) derivedfrom the coding sequence of SEQ ID NO: 215 shown in FIG. 215.

[0320]FIG. 217 shows a nucleotide sequence (SEQ ID NO: 217) of a nativesequence PRO1325 cDNA, wherein SEQ ID NO: 217 is a clone designatedherein as “DNA66659-1593”.

[0321]FIG. 218 shows the amino acid sequence (SEQ ID NO: 218) derivedfrom the coding sequence of SEQ ID NO: 217 shown in FIG. 217.

[0322]FIG. 219 shows a nucleotide sequence (SEQ ID NO: 219) of a nativesequence PRO1329 cDNA, wherein SEQ ID NO: 219 is a clone designatedherein as “DNA66660-1585”.

[0323]FIG. 220 shows the amino acid sequence (SEQ ID NO: 220) derivedfrom the coding sequence of SEQ ID NO: 219 shown in FIG. 219.

[0324]FIG. 221 shows a nucleotide sequence (SEQ ID NO: 221) of a nativesequence PRO1338 cDNA, wherein SEQ ID NO: 221 is a clone designatedherein as “DNA66667-1596”.

[0325]FIG. 222 shows the amino acid sequence (SEQ ID NO: 222) derivedfrom the coding sequence of SEQ ID NO: 221 shown in FIG. 221.

[0326]FIG. 223 shows a nucleotide sequence (SEQ ID NO: 223) of a nativesequence PRO1337 cDNA, wherein SEQ ID NO: 223 is a clone designatedherein as “DNA66672-1586”.

[0327]FIG. 224 shows the amino acid sequence (SEQ ID NO: 224) derivedfrom the coding sequence of SEQ ID NO: 223 shown in FIG. 223.

[0328]FIG. 225 shows a nucleotide sequence (SEQ ID NO: 225) of a nativesequence PRO1343 cDNA, wherein SEQ ID NO: 225 is a clone designatedherein as “DNA66675-1587”.

[0329]FIG. 226 shows the amino acid sequence (SEQ ID NO: 226) derivedfrom the coding sequence of SEQ ID NO: 225 shown in FIG. 225.

[0330]FIG. 227 shows a nucleotide sequence (SEQ ID NO: 227) of a nativesequence PRO1376 cDNA, wherein SEQ ID NO: 227 is a clone designatedherein as “DNA67300-1605”.

[0331]FIG. 228 shows the amino acid sequence (SEQ ID NO: 228) derivedfrom the coding sequence of SEQ ID NO: 227 shown in FIG. 227.

[0332]FIG. 229 shows a nucleotide sequence (SEQ ID NO: 229) of a nativesequence PRO1434 cDNA, wherein SEQ ID NO: 229 is a clone designatedherein as “DNA68818-2536”.

[0333]FIG. 230 shows the amino acid sequence (SEQ ID NO: 230) derivedfrom the coding sequence of SEQ ID NO: 229 shown in FIG. 229.

[0334]FIG. 231 shows a nucleotide sequence (SEQ ID NO: 231) of a nativesequence PRO3579 cDNA, wherein SEQ ID NO: 231 is a clone designatedherein as “DNA68862-2546”.

[0335]FIG. 232 shows the amino acid sequence (SEQ ID NO: 232) derivedfrom the coding sequence of SEQ ID NO: 231 shown in FIG. 231.

[0336]FIG. 233 shows a nucleotide sequence (SEQ ID NO: 233) of a nativesequence PRO1387 cDNA, wherein SEQ ID NO: 233 is a clone designatedherein as “DNA68872-1620”.

[0337]FIG. 234 shows the amino acid sequence (SEQ ID NO: 234) derivedfrom the coding sequence of SEQ ID NO: 233 shown in FIG. 233.

[0338]FIG. 235 shows a nucleotide sequence (SEQ ID NO: 235) of a nativesequence PRO1419 cDNA, wherein SEQ ID NO: 235 is a clone designatedherein as “DNA71290-1630”.

[0339]FIG. 236 shows the amino acid sequence (SEQ ID NO: 236) derivedfrom the coding sequence of SEQ ID NO: 235 shown in FIG. 235.

[0340]FIG. 237 shows a nucleotide sequence (SEQ ID NO: 237) of a nativesequence PRO1488 cDNA, wherein SEQ ID NO: 237 is a clone designatedherein as “DNA73736-1657”.

[0341]FIG. 238 shows the amino acid sequence (SEQ ID NO: 238) derivedfrom the coding sequence of SEQ ID NO: 237 shown in FIG. 237.

[0342]FIG. 239 shows a nucleotide sequence (SEQ ID NO: 239) of a nativesequence PRO1474 cDNA, wherein SEQ ID NO: 239 is a clone designatedherein as “DNA73739-1645”.

[0343]FIG. 240 shows the amino acid sequence (SEQ ID NO: 240) derivedfrom the coding sequence of SEQ ID NO: 239 shown in FIG. 239.

[0344]FIG. 241 shows a nucleotide sequence (SEQ ID NO: 241) of a nativesequence PRO1508 cDNA, wherein SEQ ID NO: 241 is a clone designatedherein as “DNA73742-1662”.

[0345]FIG. 242 shows the amino acid sequence (SEQ ID NO: 242) derivedfrom the coding sequence of SEQ ID NO: 241 shown in FIG. 241.

[0346]FIG. 243 shows a nucleotide sequence (SEQ ID NO: 243) of a nativesequence PRO1754 cDNA, wherein SEQ ID NO: 243 is a clone designatedherein as “DNA76385-1692”.

[0347]FIG. 244 shows the amino acid sequence (SEQ ID NO: 244) derivedfrom the coding sequence of SEQ ID NO: 243 shown in FIG. 243.

[0348]FIG. 245 shows a nucleotide sequence (SEQ ID NO: 245) of a nativesequence PRO1550 cDNA, wherein SEQ ID NO: 245 is a clone designatedherein as “DNA76393-1664”.

[0349]FIG. 246 shows the amino acid sequence (SEQ ID NO: 246) derivedfrom the coding sequence of SEQ ID NO: 245 shown in FIG. 245.

[0350]FIG. 247 shows a nucleotide sequence (SEQ ID NO: 247) of a nativesequence PRO1758 cDNA, wherein SEQ ID NO: 247 is a clone designatedherein as “DNA76399-1700”.

[0351]FIG. 248 shows the amino acid sequence (SEQ ID NO: 248) derivedfrom the coding sequence of SEQ ID NO: 247 shown in FIG. 247.

[0352]FIG. 249 shows a nucleotide sequence (SEQ ID NO: 249) of a nativesequence PRO1917 cDNA, wherein SEQ ID NO: 249 is a clone designatedherein as “DNA76400-2528”.

[0353]FIG. 250 shows the amino acid sequence (SEQ ID NO: 250) derivedfrom the coding sequence of SEQ ID NO: 249 shown in FIG. 249.

[0354]FIG. 251 shows a nucleotide sequence (SEQ ID NO: 251) of a nativesequence PRO1787 cDNA, wherein SEQ ID NO: 251 is a clone designatedherein as “DNA76510-2504”.

[0355]FIG. 252 shows the amino acid sequence (SEQ ID NO: 252) derivedfrom the coding sequence of SEQ ID NO: 251 shown in FIG. 251.

[0356]FIG. 253 shows a nucleotide sequence (SEQ ID NO: 253) of a nativesequence PRO1556 cDNA, wherein SEQ ID NO: 253 is a clone designatedherein as “DNA76529-1666”.

[0357]FIG. 254 shows the amino acid sequence (SEQ ID NO: 254) derivedfrom the coding sequence of SEQ ID NO: 253 shown in FIG. 253.

[0358]FIG. 255 shows a nucleotide sequence (SEQ ID NO: 255) of a nativesequence PRO1760 cDNA, wherein SEQ ID NO: 255 is a clone designatedherein as “DNA76532-1702”.

[0359]FIG. 256 shows the amino acid sequence (SEQ ID NO: 256) derivedfrom the coding sequence of SEQ ID NO: 255 shown in FIG. 255.

[0360]FIG. 257 shows a nucleotide sequence (SEQ ID NO: 257) of a nativesequence PRO1567 cDNA, wherein SEQ ID NO: 257 is a clone designatedherein as “DNA76541-1675”.

[0361]FIG. 258 shows the amino acid sequence (SEQ ID NO: 258) derivedfrom the coding sequence of SEQ ID NO: 257 shown in FIG. 257.

[0362]FIG. 259 shows a nucleotide sequence (SEQ ID NO: 259) of a nativesequence PRO1600 cDNA, wherein SEQ ID NO: 259 is a clone designatedherein as “DNA77503-1686”.

[0363]FIG. 260 shows the amino acid sequence (SEQ ID NO: 260) derivedfrom the coding sequence of SEQ ID NO: 259 shown in FIG. 259.

[0364]FIG. 261 shows a nucleotide sequence (SEQ ID NO: 261) of a nativesequence PRO1868 cDNA, wherein SEQ ID NO: 261 is a clone designatedherein as “DNA77624-2515”.

[0365]FIG. 262 shows the amino acid sequence (SEQ ID NO: 262) derivedfrom the coding sequence of SEQ ID NO: 261 shown in FIG. 261.

[0366]FIG. 263 shows a nucleotide sequence (SEQ ID NO: 263) of a nativesequence PRO1890 cDNA, wherein SEQ ID NO: 263 is a clone designatedherein as “DNA79230-2525”.

[0367]FIG. 264 shows the amino acid sequence (SEQ ID NO: 264) derivedfrom the coding sequence of SEQ ID NO: 263 shown in FIG. 263.

[0368]FIG. 265 shows a nucleotide sequence (SEQ ID NO: 265) of a nativesequence PRO1887 cDNA, wherein SEQ ID NO: 265 is a clone designatedherein as “DNA79862-2522”.

[0369]FIG. 266 shows the amino acid sequence (SEQ ID NO: 265) derivedfrom the coding sequence of SEQ ID NO: 265 shown in FIG. 265.

[0370]FIG. 267 shows a nucleotide sequence (SEQ ID NO: 267) of a nativesequence PRO4353 cDNA, wherein SEQ ID NO: 267 is a clone designatedherein as “DNA80145-2594”.

[0371]FIG. 268 shows the amino acid sequence (SEQ ID NO: 268) derivedfrom the coding sequence of SEQ ID NO: 267 shown in FIG. 267.

[0372]FIG. 269 shows a nucleotide sequence (SEQ ID NO: 269) of a nativesequence PRO1782 cDNA, wherein SEQ ID NO: 269 is a clone designatedherein as “DNA80899-2501”.

[0373]FIG. 270 shows the amino acid sequence (SEQ ID NO: 270) derivedfrom the coding sequence of SEQ ID NO: 269 shown in FIG. 269.

[0374]FIG. 271 shows a nucleotide sequence (SEQ ID NO: 271) of a nativesequence PRO1928 cDNA, wherein SEQ ID NO: 271 is a clone designatedherein as “DNA81754-2532”.

[0375]FIG. 272 shows the amino acid sequence (SEQ ID NO: 272) derivedfrom the coding sequence of SEQ ID NO: 271 shown in FIG. 271.

[0376]FIG. 273 shows a nucleotide sequence (SEQ ID NO: 273) of a nativesequence PRO1865 cDNA, wherein SEQ ID NO: 273 is a clone designatedherein as “DNA81757-2512”.

[0377]FIG. 274 shows the amino acid sequence (SEQ ID NO: 274) derivedfrom the coding sequence of SEQ ID NO: 273 shown in FIG. 273.

[0378]FIG. 275 shows a nucleotide sequence (SEQ ID NO: 275) of a nativesequence PRO4341 cDNA, wherein SEQ ID NO: 275 is a clone designatedherein as “DNA81761-2583”.

[0379]FIG. 276 shows the amino acid sequence (SEQ ID NO: 276) derivedfrom the coding sequence of SEQ ID NO: 275 shown in FIG. 275.

[0380]FIG. 277 shows a nucleotide sequence (SEQ ID NO: 277) of a nativesequence PRO6714 cDNA, wherein SEQ ID NO: 277 is a clone designatedherein as “DNA82358-2738”.

[0381]FIG. 278 shows the amino acid sequence (SEQ ID NO: 278) derivedfrom the coding sequence of SEQ ID NO: 277 shown in FIG. 277.

[0382]FIG. 279 shows a nucleotide sequence (SEQ ID NO: 279) of a nativesequence PRO5723 cDNA, wherein SEQ ID NO: 279 is a clone designatedherein as “DNA82361”.

[0383]FIG. 280 shows the amino acid sequence (SEQ ID NO: 280) derivedfrom the coding sequence of SEQ ID NO: 279 shown in FIG. 279.

[0384]FIG. 281 shows a nucleotide sequence (SEQ ID NO: 281) of a nativesequence PRO3438 cDNA, wherein SEQ ID NO: 281 is a clone designatedherein as “DNA82364-2538”.

[0385]FIG. 282 shows the amino acid sequence (SEQ ID NO: 282) derivedfrom the coding sequence of SEQ ID NO: 281 shown in FIG. 281.

[0386]FIG. 283 shows a nucleotide sequence (SEQ ID NO: 283) of a nativesequence PRO6071 cDNA, wherein SEQ ID NO: 283 is a clone designatedherein as “DNA82403-2959”.

[0387]FIG. 284 shows the amino acid sequence (SEQ ID NO: 284) derivedfrom the coding sequence of SEQ ID NO: 283 shown in FIG. 283.

[0388]FIG. 285 shows a nucleotide sequence (SEQ ID NO: 285) of a nativesequence PRO1801 cDNA, wherein SEQ ID NO: 285 is a clone designatedherein as “DNA83500-2506”.

[0389]FIG. 286 shows the amino acid sequence (SEQ ID NO: 286) derivedfrom the coding sequence of SEQ ID NO: 285 shown in FIG. 285.

[0390]FIG. 287 shows a nucleotide sequence (SEQ ID NO: 287) of a nativesequence PRO4324 cDNA, wherein SEQ ID NO: 287 is a clone designatedherein as “DNA83560-2569”.

[0391]FIG. 288 shows the amino acid sequence (SEQ ID NO: 288) derivedfrom the coding sequence of SEQ ID NO: 287 shown in FIG. 287.

[0392]FIG. 289 shows a nucleotide sequence (SEQ ID NO: 289) of a nativesequence PRO4333 cDNA, wherein SEQ ID NO: 289 is a clone designatedherein as “DNA84210-2576”.

[0393]FIG. 290 shows the amino acid sequence (SEQ ID NO: 290) derivedfrom the coding sequence of SEQ ID NO: 289 shown in FIG. 289.

[0394]FIG. 291 shows a nucleotide sequence (SEQ ID NO: 291) of a nativesequence PRO4405 cDNA, wherein SEQ ID NO: 291 is a clone designatedherein as “DNA84920-2614”.

[0395]FIG. 292 shows the amino acid sequence (SEQ ID NO: 292) derivedfrom the coding sequence of SEQ ID NO: 291 shown in FIG. 291.

[0396]FIG. 293 shows a nucleotide sequence (SEQ ID NO: 293) of a nativesequence PRO4356 cDNA, wherein SEQ ID NO: 293 is a clone designatedherein as “DNA86576-2595”.

[0397]FIG. 294 shows the amino acid sequence (SEQ ID NO: 294) derivedfrom the coding sequence of SEQ ID NO: 293 shown in FIG. 293.

[0398]FIG. 295 shows a nucleotide sequence (SEQ ID NO: 295) of a nativesequence PRO3444 cDNA, wherein SEQ ID NO: 295 is a clone designatedherein as “DNA87997”.

[0399]FIG. 296 shows the amino acid sequence (SEQ ID NO: 296) derivedfrom the coding sequence of SEQ ID NO: 295 shown in FIG. 295.

[0400]FIG. 297 shows a nucleotide sequence (SEQ ID NO: 297) of a nativesequence PRO4302 cDNA, wherein SEQ ID NO: 297 is a clone designatedherein as “DNA92218-2554”.

[0401]FIG. 298 shows the amino acid sequence (SEQ ID NO: 298) derivedfrom the coding sequence of SEQ ID NO: 297 shown in FIG. 297.

[0402]FIG. 299 shows a nucleotide sequence (SEQ ID NO: 299) of a nativesequence PRO4371 cDNA, wherein SEQ ID NO: 299 is a clone designatedherein as “DNA92233-2599”.

[0403]FIG. 300 shows the amino acid sequence (SEQ ID NO: 300) derivedfrom the coding sequence of SEQ ID NO: 299 shown in FIG. 299.

[0404]FIG. 301 shows a nucleotide sequence (SEQ ID NO: 301) of a nativesequence PRO4354 cDNA, wherein SEQ ID NO: 301 is a clone designatedherein as “DNA92256-2596”.

[0405]FIG. 302 shows the amino acid sequence (SEQ ID NO: 302) derivedfrom the coding sequence of SEQ ID NO: 301 shown in FIG. 301.

[0406]FIG. 303 shows a nucleotide sequence (SEQ ID NO: 303) of a nativesequence PRO5725 cDNA, wherein SEQ ID NO: 303 is a clone designatedherein as “DNA92265-2669”.

[0407]FIG. 304 shows the amino acid sequence (SEQ ID NO: 304) derivedfrom the coding sequence of SEQ ID NO: 303 shown in FIG. 303.

[0408]FIG. 305 shows a nucleotide sequence (SEQ ID NO: 305) of a nativesequence PRO4408 cDNA, wherein SEQ ID NO: 305 is a clone designatedherein as “DNA92274-2617”.

[0409]FIG. 306 shows the amino acid sequence (SEQ ID NO: 306) derivedfrom the coding sequence of SEQ ID NO: 305 shown in FIG. 305.

[0410]FIG. 307 shows a nucleotide sequence (SEQ ID NO: 307) of a nativesequence PRO9940 cDNA, wherein SEQ ID NO: 307 is a clone designatedherein as “DNA92282”.

[0411]FIG. 308 shows the amino acid sequence (SEQ ID NO: 308) derivedfrom the coding sequence of SEQ ID NO: 307 shown in FIG. 307.

[0412]FIG. 309 shows a nucleotide sequence (SEQ ID NO: 309) of a nativesequence PRO5737 cDNA, wherein SEQ ID NO: 309 is a clone designatedherein as “DNA92929-2534-1”.

[0413]FIG. 310 shows the amino acid sequence (SEQ ID NO: 310) derivedfrom the coding sequence of SEQ ID NO: 309 shown in FIG. 309.

[0414]FIG. 311 shows a nucleotide sequence (SEQ ID NO: 311) of a nativesequence PRO4425 cDNA, wherein SEQ ID NO: 311 is a clone designatedherein as “DNA93011-2637”.

[0415]FIG. 312 shows the amino acid sequence (SEQ ID NO: 312) derivedfrom the coding sequence of SEQ ID NO: 311 shown in FIG. 311.

[0416]FIG. 313 shows a nucleotide sequence(SEQ ID NO: 313) of a nativesequence PRO4345 cDNA, wherein SEQ ID NO: 313 is a clone designatedherein as “DNA94854-2586”.

[0417]FIG. 314 shows the amino acid sequence (SEQ ID NO: 314) derivedfrom the coding sequence of SEQ ID NO: 313 shown in FIG. 313.

[0418]FIG. 315 shows a nucleotide sequence (SEQ ID NO: 315) of a nativesequence PRO4342 cDNA, wherein SEQ ID NO: 315 is a clone designatedherein as “DNA96787-2534-1”.

[0419]FIG. 316 shows the amino acid sequence (SEQ ID NO: 316) derivedfrom the coding sequence of SEQ ID NO: 315 shown in FIG. 315.

[0420]FIG. 317 shows a nucleotide sequence (SEQ ID NO: 317) of a nativesequence PRO3562 cDNA, wherein SEQ ID NO: 317 is a clone designatedherein as “DNA96791 ”.

[0421]FIG. 318 shows the amino acid sequence (SEQ ID NO: 318) derivedfrom the coding sequence of SEQ ID NO: 317 shown in FIG. 317.

[0422]FIG. 319 shows a nucleotide sequence (SEQ ID NO: 319) of a nativesequence PRO4422 cDNA, wherein SEQ ID NO: 319 is a clone designatedherein as “DNA96867-2620”.

[0423]FIG. 320 shows the amino acid sequence (SEQ ID NO: 320) derivedfrom the coding sequence of SEQ ID NO: 319 shown in FIG. 319.

[0424]FIG. 321 shows a nucleotide sequence (SEQ ID NO: 321) of a nativesequence PRO5776 cDNA, wherein SEQ ID NO: 321 is a clone designatedherein as “DNA96872-2674”.

[0425]FIG. 322 shows the amino acid sequence (SEQ ID NO: 322) derivedfrom the coding sequence of SEQ ID NO: 321 shown in FIG. 321.

[0426]FIG. 323 shows a nucleotide sequence (SEQ ID NO: 323) of a nativesequence PRO4430 cDNA, wherein SEQ ID NO: 323 is a clone designatedherein as “DNA96878-2626”.

[0427]FIG. 324 shows the amino acid sequence (SEQ ID NO: 324) derivedfrom the coding sequence of SEQ ID NO: 323 shown in FIG. 323.

[0428]FIG. 325 shows a nucleotide sequence (SEQ ID NO: 325) of a nativesequence PRO4499 cDNA, wherein SEQ ID NO: 325 is a clone designatedherein as “DNA96889-2641”.

[0429]FIG. 326 shows the amino acid sequence (SEQ ID NO: 326) derivedfrom the coding sequence of SEQ ID NO: 325 shown in FIG. 325.

[0430]FIG. 327 shows a nucleotide sequence (SEQ ID NO: 327) of a nativesequence PRO4503 cDNA, wherein SEQ ID NO: 327 is a clone designatedherein as “DNA100312-2645”.

[0431]FIG. 328 shows the amino acid sequence (SEQ ID NO: 328) derivedfrom the coding sequence of SEQ ID NO: 327 shown in FIG. 327.

[0432]FIG. 329 shows a nucleotide sequence (SEQ ID NO: 329) of a nativesequence PRO10008 cDNA, wherein SEQ ID NO: 329 is a clone designatedherein as “DNA101921”.

[0433]FIG. 330 shows the amino acid sequence (SEQ ID NO: 330) derivedfrom the coding sequence of SEQ ID NO: 329 shown in FIG. 329.

[0434]FIG. 331 shows a nucleotide sequence (SEQ ID NO: 331) of a nativesequence PRO5730 cDNA, wherein SEQ ID NO: 331 is a clone designatedherein as “DNA101926”.

[0435]FIG. 332 shows the amino acid sequence (SEQ ID NO: 332) derivedfrom the coding sequence of SEQ ID NO: 331 shown in FIG. 331.

[0436]FIG. 333 shows a nucleotide sequence (SEQ ID NO: 333) of a nativesequence PRO6008 cDNA, wherein SEQ ID NO: 333 is a clone designatedherein as “DNA102844”.

[0437]FIG. 334 shows the amino acid sequence (SEQ ID NO: 334) derivedfrom the coding sequence of SEQ ID NO: 333 shown in FIG. 333.

[0438]FIG. 335 shows a nucleotide sequence (SEQ ID NO: 335) of a nativesequence PRO4527 cDNA, wherein SEQ ID NO: 335 is a clone designatedherein as “DNA103197”.

[0439]FIG. 336 shows the amino acid sequence (SEQ ID NO: 336) derivedfrom the coding sequence of SEQ ID NO: 335 shown in FIG. 335.

[0440]FIG. 337 shows a nucleotide sequence (SEQ ID NO: 337) of a nativesequence PRO4538 cDNA, wherein SEQ ID NO: 337 is a clone designatedherein as “DNA103208”.

[0441]FIG. 338 shows the amino acid sequence (SEQ ID NO: 338) derivedfrom the coding sequence of SEQ ID NO: 337 shown in FIG. 337.

[0442]FIG. 339 shows a nucleotide sequence (SEQ ID NO: 339) of a nativesequence PRO4553 cDNA, wherein SEQ ID NO: 339 is a clone designatedherein as “DNA103223”.

[0443]FIG. 340 shows the amino acid sequence (SEQ ID NO: 340) derivedfrom the coding sequence of SEQ ID NO: 339 shown in FIG. 339.

[0444]FIG. 341 shows a nucleotide sequence (SEQ ID NO: 341) of a nativesequence PRO6006 cDNA, wherein SEQ ID NO: 341 is a clone designatedherein as “DNA105782-2693”.

[0445]FIG. 342 shows the amino acid sequence (SEQ ID NO: 342) derivedfrom the coding sequence of SEQ ID NO: 341 shown in FIG. 341.

[0446]FIG. 343 shows a nucleotide sequence (SEQ ID NO: 343) of a nativesequence PRO6029 cDNA, wherein SEQ ID NO: 343 is a clone designatedherein as “DNA105849-2704”.

[0447]FIG. 344 shows the amino acid sequence (SEQ ID NO: 344) derivedfrom the coding sequence of SEQ ID NO: 343 shown in FIG. 343.

[0448]FIG. 345 shows a nucleotide sequence (SEQ ID NO: 345) of a nativesequence PRO9821 cDNA, wherein SEQ ID NO: 345 is a clone designatedherein as “DNA108725-2766”.

[0449]FIG. 346 shows the amino acid sequence (SEQ ID NO: 346) derivedfrom the coding sequence of SEQ ID NO: 345 shown in FIG. 345.

[0450]FIG. 347 shows a nucleotide sequence (SEQ ID NO: 347) of a nativesequence PRO9820 cDNA, wherein SEQ ID NO: 347 is a clone designatedherein as “DNA108769-2765”.

[0451]FIG. 348 shows the amino acid sequence (SEQ ID NO: 348) derivedfrom the coding sequence of SEQ ID NO: 347 shown in FIG. 347.

[0452]FIG. 349 shows a nucleotide sequence (SEQ ID NO: 349) of a nativesequence PRO9771 cDNA, wherein SEQ ID NO: 349 is a clone designatedherein as “DNA119498-2965”.

[0453]FIG. 350 shows the amino acid sequence (SEQ ID NO: 350) derivedfrom the coding sequence of SEQ ID NO: 349 shown in FIG. 349.

[0454]FIG. 351 shows a nucleotide sequence (SEQ ID NO: 351) of a nativesequence PRO7436cDNA, wherein SEQ ID NO: 351 is a clone designatedherein as “DNA 19535-2756”.

[0455]FIG. 352 shows the amino acid sequence (SEQ ID NO: 352) derivedfrom the coding sequence of SEQ ID NO: 351 shown in FIG. 351.

[0456]FIG. 353 shows a nucleotide sequence (SEQ ID NO: 353) of a nativesequence PRO10096 cDNA, wherein SEQ ID NO: 353 is a clone designatedherein as “DNA125185-2806”.

[0457]FIG. 354 shows the amino acid sequence (SEQ ID NO: 354) derivedfrom the coding sequence of SEQ ID NO: 353 shown in FIG. 353.

[0458]FIG. 355 shows a nucleotide sequence (SEQ ID NO: 355) of a nativesequence PRO19670 cDNA, wherein SEQ ID NO: 355 is a clone designatedherein as “DNA131639-2874”.

[0459]FIG. 356 shows the amino acid sequence (SEQ ID NO: 356) derivedfrom the coding sequence of SEQ ID NO: 355 shown in FIG. 355.

[0460]FIG. 357 shows a nucleotide sequence (SEQ ID NO: 357) of a nativesequence PRO20044 cDNA, wherein SEQ ID NO: 357 is a clone designatedherein as “DNA139623-2893”.

[0461]FIG. 358 shows the amino acid sequence (SEQ ID NO: 358) derivedfrom the coding sequence of SEQ ID NO: 357 shown in FIG. 357.

[0462]FIG. 359 shows a nucleotide sequence (SEQ ID NO: 359) of a nativesequence PRO9873 cDNA, wherein SEQ ID NO: 359 is a clone designatedherein as “DNA143076-2787”.

[0463]FIG. 360 shows the amino acid sequence (SEQ ID NO: 360) derivedfrom the coding sequence of SEQ ID NO: 359 shown in FIG. 359.

[0464]FIG. 361 shows a nucleotide sequence (SEQ ID NO: 361) of a nativesequence PRO21366 cDNA, wherein SEQ ID NO: 361 is a clone designatedherein as “DNA143276-2975”.

[0465]FIG. 362 shows the amino acid sequence (SEQ ID NO: 362) derivedfrom the coding sequence of SEQ ID NO: 361 shown in FIG. 361.

[0466]FIG. 363 shows a nucleotide sequence (SEQ ID NO: 363) of a nativesequence PRO20040 cDNA, wherein SEQ ID NO: 363 is a clone designatedherein as “DNA164625-2890”.

[0467]FIG. 364 shows the amino acid sequence (SEQ ID NO: 364) derivedfrom the coding sequence of SEQ ID NO: 363 shown in FIG. 363.

[0468]FIG. 365 shows a nucleotide sequence (SEQ ID NO: 365) of a nativesequence PRO21184 cDNA, wherein SEQ ID NO: 365 is a clone designatedherein as “DNA167678-2963”.

[0469]FIG. 366 shows the amino acid sequence (SEQ ID NO: 366) derivedfrom the coding sequence of SEQ ID NO: 365 shown in FIG. 365.

[0470]FIG. 367 shows a nucleotide sequence (SEQ ID NO: 367) of a nativesequence PRO21055 cDNA, wherein SEQ ID NO: 367 is a clone designatedherein as “DNA170021-2923”.

[0471]FIG. 368 shows the amino acid sequence (SEQ ID NO: 368) derivedfrom the coding sequence of SEQ ID NO: 367 shown in FIG. 367.

[0472]FIG. 369 shows a nucleotide sequence (SEQ ID NO: 369) of a nativesequence PRO28631 cDNA, wherein SEQ ID NO: 369 is a clone designatedherein as “DNA170212-3000”.

[0473]FIG. 370 shows the amino acid sequence (SEQ ID NO: 370) derivedfrom the coding sequence of SEQ ID NO: 369 shown in FIG. 369.

[0474]FIG. 371 shows a nucleotide sequence (SEQ ID NO: 371) of a nativesequence PRO21384 cDNA, wherein SEQ ID NO: 371 is a clone designatedherein as “DNA177313-2982”.

[0475]FIG. 372 shows the amino acid sequence (SEQ ID NO: 372) derivedfrom the coding sequence of SEQ ID NO: 371 shown in FIG. 371.

[0476]FIG. 373 shows a nucleotide sequence (SEQ ID NO: 373) of a nativesequence PRO1449 cDNA, wherein SEQ ID NO: 373 is a clone designatedherein as “DNA64908-1163-1”.

[0477]FIG. 374 shows the amino acid sequence (SEQ ID NO: 374) derivedfrom the coding sequence of SEQ ID NO: 373 shown in FIG. 373.

[0478]FIG. 375 shows wholemount in situ hybridization result on mouseembryos using a mouse orthologue of PRO1449 which has about 78% aminoacid identity with PRO1449. The results show that PRO1449 orthologue isexpressed in the developing vasculature. The cross-section further showsexpression in endothelial cells and progenitors of endothelial cells.

[0479]FIG. 376 shows that a PRO1449 orthologue having about 78% aminoacid identity with PRO1449 is expressed in vasculature of many inflamedand diseased tissues, but is very low, or lacking, in normal adultvessels.

[0480]FIG. 377 shows that a PRO1449 orthologue having about 78% aminoacid identity with PRO1449 induces ectopic vessels in the eyes ofchicken embryos.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Definitions

[0481] The phrases “cardiovascular, endothelial and angiogenicdisorder”, “cardiovascular, endothelial and angiogenic dysfunction”,“cardiovascular, endothelial or angiogenic disorder” and“cardiovascular, endothelial or angiogenic dysfunction” are usedinterchangeably and refer in part to systemic disorders that affectvessels, such as diabetes mellitus, as well as diseases of the vesselsthemselves, such as of the arteries, capillaries, veins, and/orlymphatics. This would include indications that stimulate angiogenesisand/or cardiovascularization, and those that inhibit angiogenesis and/orcardiovascularization. Such disorders include, for example, arterialdisease, such as atherosclerosis, hypertension, inflammatoryvasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; and other vasculardisorders such as peripheral vascular disease, cancer such as vasculartumors, e.g., hemangioma (capillary and cavernous), glomus tumors,telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma, tumor angiogenesis, trauma such as wounds, burns, andother injured tissue, implant fixation, scarring, ischemia reperfusioninjury, rheumatoid arthritis, cerebrovascular disease, renal diseasessuch as acute renal failure, and osteoporosis. This would also includeangina, myocardial infarctions such as acute myocardial infarctions,cardiac hypertrophy, and heart failure such as CHF.

[0482] “Hypertrophy”, as used herein, is defined as an increase in massof an organ or structure independent of natural growth that does notinvolve tumor formation. Hypertrophy of an organ or tissue is due eitherto an increase in the mass of the individual cells (true hypertrophy),or to an increase in the number of cells making up the tissue(hyperplasia), or both. Certain organs, such as the heart, lose theability to divide shortly after birth. Accordingly, “cardiachypertrophy” is defined as an increase in mass of the heart, which, inadults, is characterized by an increase in myocyte cell size andcontractile protein content without concomitant cell division. Thecharacter of the stress responsible for inciting the hypertrophy, (e.g.,increased preload, increased afterload, loss of myocytes, as inmyocardial infarction, or primary depression of contractility), appearsto play a critical role in determining the nature of the response. Theearly stage of cardiac hypertrophy is usually characterizedmorphologically by increases in the size of myofibrils and mitochondria,as well as by enlargement of mitochondria and nuclei. At this stage,while muscle cells are larger than normal, cellular organization islargely preserved. At a more advanced stage of cardiac hypertrophy,there are preferential increases in the size or number of specificorganelles, such as mitochondria, and new contractile elements are addedin localized areas of the cells, in an irregular manner. Cells subjectedto long-standing hypertrophy show more obvious disruptions in cellularorganization, including markedly enlarged nuclei with highly lobulatedmembranes, which displace adjacent myofibrils and cause breakdown ofnormal Z-band registration. The phrase “cardiac hypertrophy” is used toinclude all stages of the progression of this condition, characterizedby various degrees of structural damage of the heart muscle, regardlessof the underlying cardiac disorder. Hence, the term also includesphysiological conditions instrumental in the development of cardiachypertrophy, such as elevated blood pressure, aortic stenosis, ormyocardial infarction.

[0483] “Heart failure” refers to an abnormality of cardiac functionwhere the heart does not pump blood at the rate needed for therequirements of metabolizing tissues. The heart failure can be caused bya number of factors, including ischemic, congenital, rheumatic, oridiopathic forms.

[0484] “Congestive heart failure” (CHF) is a progressive pathologicstate where the heart is increasingly unable to supply adequate cardiacoutput (the volume of blood pumped by the heart over time) to deliverthe oxygenated blood to peripheral tissues. As CHF progresses,structural and hemodynamic damages occur. While these damages have avariety of manifestations, one characteristic symptom is ventricularhypertrophy. CHF is a common end result of a number of various cardiacdisorders.

[0485] “Myocardial infarction” generally results from atherosclerosis ofthe coronary arteries, often with superimposed coronary thrombosis. Itmay be divided into two major types: transmural infarcts, in whichmyocardial necrosis involves the full thickness of the ventricular wall,and subendocardial (nontransmural) infarcts, in which the necrosisinvolves the subendocardium, the intramural myocardium, or both, withoutextending all the way through the ventricular wall to the epicardium.Myocardial infarction is known to cause both a change in hemodynamiceffects and an alteration in structure in the damaged and healthy zonesof the heart. Thus, for example, myocardial infarction reduces themaximum cardiac output and the stroke volume of the heart. Alsoassociated with myocardial infarction is a stimulation of the DNAsynthesis occurring in the interstice as well as an increase in theformation of collagen in the areas of the heart not affected.

[0486] As a result of the increased stress or strain placed on the heartin prolonged hypertension due, for example, to the increased totalperipheral resistance, cardiac hypertrophy has long been associated with“hypertension”. A characteristic of the ventricle that becomeshypertrophic as a result of chronic pressure overload is an impaireddiastolic performance. Fouad et al., J. Am. Coll. Cardiol., 4: 1500-1506(1984); Smith et al., J. Am. Coll. Cardiol., 5: 869-874 (1985). Aprolonged left ventricular relaxation has been detected in earlyessential hypertension, in spite of normal or supra normal systolicfunction. Hartford et al., Hypertension, 6: 329-338 (1984). However,there is no close parallelism between blood pressure levels and cardiachypertrophy. Although improvement in left ventricular function inresponse to antihypertensive therapy has been reported in humans,patients variously treated with a diuretic (hydrochlorothiazide), aβ-blocker (propranolol), or a calcium channel blocker (diltiazem), haveshown reversal of left ventricular hypertrophy, without improvement indiastolic function. Inouye et al., Am. J. Cardiol., 53: 1583-7 (1984).

[0487] Another complex cardiac disease associated with cardiachypertrophy is “hypertrophic cardiomyopathy”. This condition ischaracterized by a great diversity of morphologic, functional, andclinical features (Maron et al., N. Engl. J. Med., 316: 780-789 (1987);Spirito et al., N. Engl. J. Med., 320: 749-755 (1989); Louie andEdwards, Prog. Cardiovasc. Dis., 36: 275-308 (1994); Wigle et al.,Circulation, 92: 1680-1692 (1995)), the heterogeneity of which isaccentuated by the fact that it afflicts patients of all ages. Spiritoet al., N. Engl. J. Med., 336: 775-785 (1997). The causative factors ofhypertrophic cardiomyopathy are also diverse and little understood. Ingeneral, mutations in genes encoding sarcomeric proteins are associatedwith hypertrophic cardiomyopathy. Recent data suggest that β-myosinheavy chain mutations may account for approximately 30 to 40 percent ofcases of familial hypertrophic cardiomyopathy. Watkins et al., N. Engl.J. Med., 326: 1108-1114(1992); Schwartz et al, Circulation, 91: 532-540(1995); Marian and Roberts, Circulation, 92: 1336-1347 (1995);Thierfelder et al., Cell, 77: 701-712 (1994); Watkins et al., Nat. Gen.,11: 434-437 (1995). Besides β-myosin heavy chain, other locations ofgenetic mutations include cardiac troponin T, alpha topomyosin, cardiacmyosin binding protein C, essential myosin light chain, and regulatorymyosin light chain. See, Malik and Watkins, Curr. Opin. Cardiol., 12:295-302 (1997).

[0488] Supravalvular “aortic stenosis” is an inherited vascular disordercharacterized by narrowing of the ascending aorta, but other arteries,including the pulmonary arteries, may also be affected. Untreated aorticstenosis may lead to increased intracardiac pressure resulting inmyocardial hypertrophy and eventually heart failure and death. Thepathogenesis of this disorder is not fully understood, but hypertrophyand possibly hyperplasia of medial smooth muscle are prominent featuresof this disorder. It has been reported that molecular variants of theelastin gene are involved in the development and pathogenesis of aorticstenosis. U.S. Pat. No. 5,650,282 issued Jul. 22, 1997.

[0489] “Valvular regurgitation” occurs as a result of heart diseasesresulting in disorders of the cardiac valves. Various diseases, likerheumatic fever, can cause the shrinking or pulling apart of the valveorifice, while other diseases may result in endocarditis, aninflammation of the endocardium or lining membrane of theatrioventricular orifices and operation of the heart. Defects such asthe narrowing of the valve stenosis or the defective closing of thevalve result in an accumulation of blood in the heart cavity orregurgitation of blood past the valve. If uncorrected, prolongedvalvular stenosis or insufficiency may result in cardiac hypertrophy andassociated damage to the heart muscle, which may eventually necessitatevalve replacement.

[0490] The treatment of all these, and other cardiovascular, endothelialand angiogenic disorders, which may or may not be accompanied by cardiachypertrophy, is encompassed by the present invention.

[0491] The terms “cancer”, “cancerous”, and “malignant” refer to ordescribe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. Examples of cancer include butare not limited to, carcinoma including adenocarcinoma, lymphoma,blastoma, melanoma, sarcoma, and leukemia. More particular examples ofsuch cancers include squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, Hodgkin's andnon-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer such as hepatic carcinoma andhepatoma, bladder cancer, breast cancer, colon cancer, colorectalcancer, endometrial carcinoma, salivary gland carcinoma, kidney cancersuch as renal cell carcinoma and Wilms' tumors, basal cell carcinoma,melanoma, prostate cancer, vulval cancer, thyroid cancer, testicularcancer, esophageal cancer, and various types of head and neck cancer.The preferred cancers for treatment herein are breast, colon, lung,melanoma, ovarian, and others involving vascular tumors as noted above.

[0492] The term “cytotoxic agent” as used herein refers to a substancethat inhibits or prevents the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g., ¹³¹I, ¹²⁵I, ⁹⁰Y, and ¹⁸⁶Re), chemotherapeutic agents,and toxins such as enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof.

[0493] A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents, folic acid antagonists, anti-metabolites of nucleicacid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil,cisplatin, purine nucleosides, amines, amino acids, triazol nucleosides,or corticosteroids. Specific examples include Adriamycin, Doxorubicin,5-Fluorouracil, Cytosine arabinoside (“Ara-C”), Cyclophosphamide,Thiotepa, Busulfan, Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin,Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan, and other relatednitrogen mustards. Also included in this definition are hormonal agentsthat act to regulate or inhibit hormone action on tumors, such astamoxifen and onapristone.

[0494] A “growth-inhibitory agent” when used herein refers to a compoundor composition that inhibits growth of a cell, such as anWnt-overexpressing cancer cell, either in vitro or in vivo. Thus, thegrowth-inhibitory agent is one which significantly reduces thepercentage of malignant cells in S phase. Examples of growth-inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxol, and topo II inhibitors such as doxorubicin,daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 alsospill over into S-phase arrest, for example, DNA alkylating agents suchas tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in The Molecular Basis of Cancer, Mendelsohn and Israel, eds.,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (W B Saunders: Philadelphia,1995), especially p. 13. Additional examples include tumor necrosisfactor (TNF), an antibody capable of inhibiting or neutralizing theangiogenic activity of acidic or basic FGF or hepatocyte growth factor(HGF), an antibody capable of inhibiting or neutralizing the coagulantactivities of tissue factor, protein C, or protein S (see, WO 91/01753,published Feb. 21, 1991), or an antibody capable of binding to HER2receptor (WO 89/06692), such as the 4D5 antibody (and functionalequivalents thereof) (e.g., WO 92/22653).

[0495] “Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of acardiovascular, endothelial, and angiogenic disorder. The concept oftreatment is used in the broadest sense, and specifically includes theprevention (prophylaxis), moderation, reduction, and curing ofcardiovascular, endothelial, and angiogenic disorders of any stage.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) or ameliorate a cardiovascular, endothelial, andangiogenic disorder such as hypertrophy. Those in need of treatmentinclude those already with the disorder as well as those prone to havethe disorder or those in whom the disorder is to be prevented. Thedisorder may result from any cause, including idiopathic, cardiotrophic,or myotrophic causes, or ischemia or ischemic insults, such asmyocardial infarction.

[0496] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain theinitial effect, such as an anti-hypertrophic effect, for an extendedperiod of time.

[0497] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep,pigs, etc. Preferably, the mammal is human.

[0498] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0499] The phrase “cardiovascular, endothelial or angiogenic agents”refers generically to any drug that acts in treating cardiovascular,endothelial, and angiogenic disorders. Examples of cardiovascular agentsare those that promote vascular homeostasis by modulating bloodpressure, heart rate, heart contractility, and endothelial and smoothmuscle biology, all of which factors have a role in cardiovasculardisease. Specific examples of these include angiotensin-II receptorantagonists; endothelin receptor antagonists such as, for example,BOSENTAN™ and MOXONODIN™; interferon-gamma (IFN-γ);des-aspartate-angiotensin I; thrombolytic agents, e.g., streptokinase,urokinase, t-PA, and a t-PA variant specifically designed to have longerhalf-life and very high fibrin specificity, TNK t-PA (a T103N, N117Q,KHRR(296-299)AAAA t-PA variant, Keyt et al., Proc. Natl. Acad. Sci. USA,91: 3670-3674 (1994)); inotropic or hypertensive agents such asdigoxigenin and β-adrenergic receptor blocking agents, e g, propranolol,timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol,acetobutolol, atenolol, metoprolol, and carvedilol; angiotensinconverting enzyme (ACE) inhibitors, e.g., quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, and lisinopril; diuretics,e g., chlorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, andindapamide; and calcium channel blockers, e.g., diltiazem, nifedipine,verapamil, nicardipine. One preferred category of this type is atherapeutic agent used for the treatment of cardiac hypertrophy or of aphysiological condition instrumental in the development of cardiachypertrophy, such as elevated blood pressure, aortic stenosis, ormyocardial infarction.

[0500] “Angiogenic agents” and “endothelial agents” are active agentsthat promote angiogenesis and/or endothelial cell growth, or, ifapplicable, vasculogenesis. This would include factors that acceleratewound healing, such as growth hormone, insulin-like growth factor-I(IGF-I), VEGF, VIGF, PDGF, epidermal growth factor (EGF), CTGF andmembers of its family, FGF, and TGF-α and TGF-β.

[0501] “Angiostatic agents” are active agents that inhibit angiogenesisor vasculogenesis or otherwise inhibit or prevent growth of cancercells. Examples include antibodies or other antagonists to angiogenicagents as defined above, such as antibodies to VEGF. They additionallyinclude cytotherapeutic agents such as cytotoxic agents,chemotherapeutic agents, growth-inhibitory agents, apoptotic agents, andother agents to treat cancer, such as anti-HER-2, anti-CD20, and otherbioactive and organic chemical agents.

[0502] In a pharmacological sense, in the context of the presentinvention, a “therapeutically effective amount” of an active agent suchas a PRO polypeptide or agonist or antagonist thereto or an anti-PROantibody, refers to an amount effective in the treatment of acardiovascular, endothelial or angiogenic disorder in a mammal and canbe determined empirically.

[0503] As used herein, an “effective amount” of an active agent such asa PRO polypeptide or agonist or antagonist thereto or an anti-PROantibody, refers to an amount effective for carrying out a statedpurpose, wherein such amounts may be determined empirically for thedesired effect.

[0504] The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

[0505] A “native sequence PRO polypeptide” comprises a polypeptidehaving the same amino acid sequence as the corresponding PRO polypeptidederived from nature. Such native sequence PRO polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence PRO polypeptide” specificallyencompasses naturally-occurring truncated or secreted forms of thespecific PRO polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

[0506] The PRO polypeptide “extracellular domain” or “ECD” refers to aform of the PRO polypeptide which is essentially free of thetransmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECDwill have less than 1% of such transmembrane and/or cytoplasmic domainsand preferably, will have less than 0.5% of such domains. It will beunderstood that any transmembrane domains identified for the PROpolypeptides of the present invention are identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain as initially identified herein. Optionally, therefore, anextracellular domain of a PRO polypeptide may contain from about 5 orfewer amino acids on either side of the transmembranedomain/extracellular domain boundary as identified in the Examples orspecification and such polypeptides, with or without the associatedsignal peptide, and nucleic acid encoding them, are comtemplated by thepresent invention.

[0507] The approximate location of the “signal peptides” of the variousPRO polypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng., 10:1-6(1997) and von Heinje et al., Nucl. Acids Res., 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

[0508] “PRO polypeptide variant” means an active PRO polypeptide asdefined above or below having at least about 80% amino acid sequenceidentity with a full-length native sequence PRO polypeptide sequence asdisclosed herein, a PRO polypeptide sequence lacking the signal peptideas disclosed herein, an extracellular domain of a PRO polypeptide, withor without the signal peptide, as disclosed herein or any other fragmentof a full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80%, 81%, 82%, 83%, 84%85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%amino acid sequence identity and alternatively at least about 99% aminoacid sequence identity to a full-length native sequence PRO polypeptidesequence as disclosed herein, a PRO polypeptide sequence lacking thesignal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal peptide, as disclosed herein orany other specifically defined fragment of a full-length PRO polypeptidesequence as disclosed herein. Ordinarily, PRO variant polypeptides areat least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 aminoacids in length and alternatively at least about 300 amino acids inlength, or more.

[0509] “Percent (%) amino acid sequence identity” with respect to thePRO polypeptide sequences identified herein is defined as the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues in a PRO sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity. Alignment for purposes of determiningpercent amino acid sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using publiclyavailable computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full-length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are obtained as described below by using thesequence comparison computer program ALIGN-2, wherein the completesource code for the ALIGN-2 program is provided in Table 1. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc.,and the source code shown in Table 1 has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1. The ALIGN-2 program should be compiled for use on a UNIXoperating system, preferably digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary.

[0510] For purposes herein, the % amino acid sequence identity of agiven amino acid sequence A to, with, or against a given amino acidsequence B (which can alternatively be phrased as a given amino acidsequence A that has or comprises a certain % amino acid sequenceidentity to, with, or against a given amino acid sequence B) iscalculated as follows:

100 times the fraction {fraction (X/Y)}

[0511] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program ALIGN-2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A. As examples of % amino acid sequenceidentity calculations, Tables 2-3 demonstrate how to calculate the %amino acid sequence identity of the amino acid sequence designated“Comparison Protein” to the amino acid sequence designated “PRO”.

[0512] Unless specifically stated otherwise, all % amino acid sequenceidentity values used herein are obtained as described above using theALIGN-2 sequence comparison computer program. However, % amino acidsequence identity may also be determined using the sequence comparisonprogram NCBI-BLAST2 (Altschul et al., Nucleic Acids Res., 25:3389-3402(1997)). The NCBI-BLAST2 sequence comparison program may be downloadedfrom http://www.ncbi.nlm.nih.gov. or otherwise obtained from theNational Institute of Health, Bethesda, Md. NCBI-BLAST2 uses severalsearch parameters, wherein all of those search parameters are set todefault values including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

[0513] In situations where NCBI-BLAST2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction {fraction (X/Y)}

[0514] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

[0515] In addition, % amino acid sequence identity may also bedetermined using the WU-BLAST-2 computer program (Altschul et al.,Methods in Enzymology, 266:460-480 (1996)). Most of the WU-BLAST-2search parameters are set to the default values. Those not set todefault values, i.e., the adjustable parameters, are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11, and scoring matrix=BLOSUM62. For purposes herein, a % amino acidsequence identity value is determined by dividing (a) the number ofmatching identical amino acids residues between the amino acid sequenceof the PRO polypeptide of interest having a sequence derived from thenative PRO polypeptide and the comparison amino acid sequence ofinterest (i.e., the sequence against which the PRO polypeptide ofinterest is being compared which may be a PRO variant polypeptide) asdetermined by WU-BLAST-2 by (b) the total number of amino acid residuesof the PRO polypeptide of interest. For example, in the statement “apolypeptide comprising an amino acid sequence A which has or having atleast 80% amino acid sequence identity to the amino acid sequence B”,the amino acid sequence A is the comparison amino acid sequence ofinterest and the amino acid sequence B is the amino acid sequence of thePRO polypeptide of interest.

[0516] “PRO variant polynucleotide” or “PRO variant nucleic acidsequence” means a nucleic acid molecule which encodes an active PROpolypeptide as defined below and which has at least about 80% nucleicacid sequence identity with a nucleotide acid sequence encoding afull-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal peptide, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Ordinarily, a PRO variant polynucleotide will have atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% or 98% nucleic acid sequence identity andalternatively at least about 99% nucleic acid sequence identity with anucleic acid sequence encoding a full-length native sequence PROpolypeptide sequence as disclosed herein, a full-length native sequencePRO polypeptide sequence lacking the signal peptide as disclosed herein,an extracellular domain of a PRO polypeptide, with or without the signalsequence, as disclosed herein or any other fragment of a full-length PROpolypeptide sequence as disclosed herein. Variants do not encompass thenative nucleotide sequence.

[0517] Ordinarily, PRO variant polynucleotides are at least about 30,60, 90, 120, 150, 180, 210, 240, 270, 300, 450, or 600 nucleotides inlength and alternatively at least about 900 nucleotides in length, ormore.

[0518] “Percent (%) nucleic acid sequence identity” with respect to thePRO polypeptide-encoding nucleic acid sequences identified herein isdefined as the percentage of nucleotides in a candidate sequence thatare identical with the nucleotides in a PRO polypeptide-encoding nucleicacid sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent nucleic acid sequence identity canbe achieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % nucleic acid sequence identity values are obtained asdescribed below by using the sequence comparison computer programALIGN-2, wherein the complete source code for the ALIGN-2 program isprovided in Table 1. The ALIGN-2 sequence comparison computer programwas authored by Genentech, Inc., and the source code shown in Table 1has been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in Table 1. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

[0519] For purposes herein, the % nucleic acid sequence identity of agiven nucleic acid sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given nucleic acidsequence C that has or comprises a certain % nucleic acid sequenceidentity to, with, or against a given nucleic acid sequence D) iscalculated as follows:

100 times the fraction {fraction (W/Z)}

[0520] where W is the number of nucleotides scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofC and D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4-5 demonstrate how to calculate the % nucleic acidsequence identity of the nucleic acid sequence designated “ComparisonDNA” to the nucleic acid sequence designated “PRO-DNA”.

[0521] Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described above using theALIGN-2 sequence comparison computer program. However, % nucleic acidsequence identity may also be determined using the sequence comparisonprogram NCBI-BLAST2 (Altschul et al., Nucleic Acids Res., 25:3389-3402(1997)). The NCBI-BLAST2 sequence comparison program may be downloadedfrom http://www.ncbi.nlm.nih.gov. or otherwise obtained from theNational Institute of Health, Bethesda, Md. NCBI-BLAST2 uses severalsearch parameters, wherein all of those search parameters are set todefault values including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

[0522] In situations where NCBI-BLAST2 is employed for sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction {fraction (W/Z)}

[0523] where W is the number of nucleotides scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of C and D, and where Z is the total number of nucleotides inD. It will be appreciated that where the length of nucleic acid sequenceC is not equal to the length of nucleic acid sequence D, the % nucleicacid sequence identity of C to D will not equal the % nucleic acidsequence identity of D to C.

[0524] In addition, % nucleic acid sequence identity values may also begenerated using the WU-BLAST-2 computer program (Altschul et al.,Methods in Enzymology, 266:460-480 (1996)). Most of the WU-BLAST-2search parameters are set to the default values. Those not set todefault values, i.e., the adjustable parameters, are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11, and scoring matrix=BLOSUM62. For purposes herein, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

[0525] In other embodiments, PRO variant polynucleotides are nucleicacid molecules that encode an active PRO polypeptide and which arecapable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding the full-length PROpolypeptide as shown in the specification herein and accompanyingfigures. PRO variant polypeptides may be those that are encoded by a PROvariant polynucleotide.

[0526] “Isolated”, when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Preferably, the isolated polypeptide is free of association with allcomponents with which it is naturally associated. Contaminant componentsof its natural environment are materials that would typically interferewith diagnostic or therapeutic uses for the polypeptide, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the polypeptide will be purified (1) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (2)to homogeneity by SDS-PAGE under non-reducing or reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated polypeptideincludes polypeptide in situ within recombinant cells, since at leastone component of the PRO natural environment will not be present.Ordinarily, however, isolated polypeptide will be prepared by at leastone purification step.

[0527] An “isolated” nucleic acid molecule encoding a PRO polypeptide oran “isolated” nucleic acid molecule encoding an anti-PRO antibody is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the PRO-encoding nucleic acid or the naturalsource of the anti-PRO-encoding nucleic acid. Preferably, the isolatednucleic acid is free of association with all components with which it isnaturally associated. An isolated PRO-encoding nucleic acid molecule oran isolated anti-PRO-encoding nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the PRO-encoding nucleic acidmolecule or from the anti-PRO-encoding nucleic acid molecule as itexists in natural cells. However, an isolated nucleic acid moleculeencoding a PRO polypeptide or an isolated nucleic acid molecule encodingan anti-PRO antibody includes PRO-nucleic acid molecules oranti-PRO-nucleic acid molecules contained in cells that ordinarilyexpress PRO polypeptides or anti-PRO antibodies where, for example, thenucleic acid molecule is in a chromosomal location different from thatof natural cells.

[0528] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize, forexample, promoters, polyadenylation signals, and enhancers.

[0529] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora PRO polypeptide if it is expressed as a preprotein that participatesin the secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inthe same reading frame. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0530] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see, Ausubel etal., Current Protocols in Molecular Biology (Wiley IntersciencePublishers, 1995).

[0531] “Stringent conditions” or “high-stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example, 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

[0532] “Moderately-stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:Cold Spring Harbor Press, 1989), and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength, and %SDS) less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

[0533] The modifier “epitope-tagged” when used herein refers to achimeric polypeptide comprising a PRO polypeptide fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the polypeptide to which itis fused. The tag polypeptide preferably also is fairly unique so thatthe antibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

[0534] “Active” or “activity” in the context of PRO variants refers toform(s) of PRO proteins that retain the biologic and/or immunologicactivities of a native or naturally-occurring PRO polypeptide.

[0535] “Biological activity” in the context of a molecule thatantagonizes a PRO polypeptide that can be identified by the screeningassays disclosed herein (e.g., an organic or inorganic small molecule,peptide, etc.) is used to refer to the ability of such molecules to bindor complex with the PRO polypeptide identified herein, or otherwiseinterfere with the interaction of the PRO polypeptide with othercellular proteins or otherwise inhibits the transcription or translationof the PRO polypeptide. Particularly preferred biological activityincludes cardiac hypertrophy, activity that acts on systemic disordersthat affect vessels, such as diabetes mellitus, as well as diseases ofthe arteries, capillaries, veins, and/or lymphatics, and cancer.

[0536] The term “antagonist” is used in the broadest sense, and includesany molecule that partially or fully blocks, inhibits, or neutralizesone or more of the biological activities of a native PRO polypeptidedisclosed herein, for example, if applicable, its mitogenic orangiogenic activity. Antagonists of a PRO polypeptide may act byinterfering with the binding of a PRO polypeptide to a cellularreceptor, by incapacitating or killing cells that have been activated bya PRO polypeptide, or by interfering with vascular endothelial cellactivation after binding of a PRO polypeptide to a cellular receptor.All such points of intervention by a PRO polypeptide antagonist shall beconsidered equivalent for purposes of this invention. The antagonistsinhibit the mitogenic, angiogenic, or other biological activity of PROpolypeptides, and thus are useful for the treatment of diseases ordisorders characterized by undesirable excessive neovascularization,including by way of example tumors, and especially solid malignanttumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic andother retinopathies, retrolental fibroplasia, age-related maculardegeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias(including Grave's disease), corneal and other tissue transplantation,and chronic inflammation. The antagonists also are useful for thetreatment of diseases or disorders characterized by undesirableexcessive vascular permeability, such as edema associated with braintumors, ascites associated with malignancies, Meigs' syndrome, lunginflammation, nephrotic syndrome, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion. In a similarmanner, the term “agonist” is used in the broadest sense and includesany molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments, or amino acid sequence variants of native PROpolypeptides, peptides, small organic molecules, etc.

[0537] A “small molecule” is defined herein to have a molecular weightbelow about 500 daltons.

[0538] The term “PRO polypeptide receptor” as used herein refers to acellular receptor for a PRO polypeptide, ordinarily a cell-surfacereceptor found on vascular endothelial cells, as well as variantsthereof that retain the ability to bind a PRO polypeptide.

[0539] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.The term “antibody” is used in the broadest sense and specificallycovers, without limitation, intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies)formed from at least two intact antibodies, and antibody fragments, solong as they exhibit the desired biological activity.

[0540] “Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

[0541] The term “variable” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody toand for its particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three segments called complementarity-determiningregions (CDRs) or hypervariable regions both in the light-chain and theheavy-chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FR). The variabledomains of native heavy and light chains each comprise four FR regions,largely adopting a β-sheet configuration, connected by three CDRs, whichform loops connecting, and in some cases forming part of, the 62 -sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen-binding site of antibodies. See, Kabat etal., NIH Publ. No. 91-3242, Vol. I, pages 647-669 (1991). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

[0542] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.,8(10): 1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0543] Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, whose name reflectsits ability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen-combining sites and is still capable ofcross-linking antigen.

[0544] “Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site.

[0545] This region consists of a dimer of one heavy- and one light-chainvariable domain in tight, non-covalent association. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody.

[0546] However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site.

[0547] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments that have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

[0548] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa (κ) and lambda (λ), based on the amino acid sequences oftheir constant domains.

[0549] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM; and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

[0550] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally-occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations that typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example,. the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., Nature, 256: 495 (1975), or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:

[0551]624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597(1991), for example.

[0552] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity. U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984).

[0553] “Humanized” forms of non-human (e.g., murine) antibodies arechimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv FR residuesof the human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues thatare found neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andmaximize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody preferably also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al, Nature, 321:522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2: 593-596 (1992). The humanizedantibody includes a PRIMATIZED™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

[0554] “Single-chain Fv” or “sFv” antibody fragments comprise the V_(H)and V_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv see, Pluckthun in The Pharmacology of MonoclonalAntibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag: NewYork, 1994), pp. 269-315.

[0555] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0556] An “isolated” antibody is one that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells, since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0557] An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

[0558] The word “label” when used herein refers to a detectable compoundor other composition that is conjugated directly or indirectly to theantibody so as to generate a “labeled” antibody. The label may bedetectable by itself (e.g., radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition that is detectable. Radionuclidesthat can serve as detectable labels include, for example, I-131, I-123,I-125, Y-90, Re-188, At-211, Cu-67, Bi-212, and Pd-109. The label mayalso be a non-detectable entity such as a toxin.

[0559] By “solid phase” is meant a non-aqueous matrix to which anantibody of the present invention can adhere.

[0560] Examples of solid phases encompassed herein include those formedpartially or entirely of glass (e.g., controlled pore glass),polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinylalcohol and silicones. In certain embodiments, depending on the context,the solid phase can comprise the well of an assay plate; in others it isa purification column (e.g., an affinity chromatography column). Thisterm also includes a discontinuous solid phase of discrete particles,such as those described in U.S. Pat. No. 4,275,149.

[0561] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant that is useful for delivery of adrug (such as the PRO polypeptide or antibodies thereto disclosedherein) to a mammal. The components of the liposome are commonlyarranged in a bilayer formation, similar to the lipid arrangement ofbiological membranes.

[0562] As used herein, the term “immunoadhesin” designates antibody-likemolecules that combine the binding specificity of a heterologous protein(an “adhesin”) with the effector functions of immunoglobulin constantdomains. Structurally, the immunoadhesins comprise a fusion of an aminoacid sequence with the desired binding specificity that is other thanthe antigen recognition and binding site of an antibody (i.e., is“heterologous”), and an immunoglobulin constant domain sequence. Theadhesin part of an immunoadhesin molecule typically is a contiguousamino acid sequence comprising at least the binding site of a receptoror a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD, or IgM.

[0563] As shown below, Table 1 provides the complete source code for theALIGN-2 sequence comparison computer program. This source code may beroutinely compiled for use on a UNIX operating system to provide theALIGN-2 sequence comparison computer program.

[0564] In addition, Tables 2-5 show hypothetical exemplifications forusing the below described method to determine % amino acid sequenceidentity (Tables 2-3) and % nucleic acid sequence identity (Tables 4-5)using the ALIGN-2 sequence comparison computer program, wherein “PRO”represents the amino acid sequence of a hypothetical PRO polypeptide ofinterest, “Comparison Protein” represents the amino acid sequence of apolypeptide against which the “PRO” polypeptide of interest is beingcompared, “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, “X”, “Y”, and “Z” eachrepresent different hypothetical amino acid residues and “N”, “L” and“V” each represent different hypothetical nucleotides.

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein

[0565] TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein

[0566] TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA

[0567] TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonDNA NNNNLLLVV (Length =  9 nucleotides)

5.2. Compositions and Methods of the Invention

[0568] 5.2.1. PRO Variants

[0569] In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO polypeptidesuch as changing the number or position of glycosylation sites oraltering the membrane anchoring characteristics.

[0570] Variations in the native full-length sequence PRO polypeptide orin various domains of the PRO polypeptide described herein, can be made,for example, using any of the techniques and guidelines for conservativeand non-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the PRO polypeptide that results in a changein the amino acid sequence of the PRO polypeptide as compared with thenative sequence PRO polypeptide. Optionally the variation is bysubstitution of at least one amino acid with any other amino acid in oneor more of the domains of the PRO polypeptide. Guidance in determiningwhich amino acid residue may be inserted, substituted or deleted withoutadversely affecting the desired activity may be found by comparing thesequence of the PRO polypeptide with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

[0571] In particular embodiments, conservative substitutions of interestare shown in Table 6 under the heading of preferred substitutions. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 6 Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) val; leu; ile valArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leunorleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg;gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyrleu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyrTyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala;norleucine

[0572] Substantial modifications in function or immunological identityof the PRO polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

[0573] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0574] (2) neutral hydrophilic: cys, ser, thr;

[0575] (3) acidic: asp, glu;

[0576] (4) basic: asn, gln, his, lys, arg;

[0577] (5) residues that influence chain orientation: gly, pro; and

[0578] (6) aromatic: trp, tyr, phe.

[0579] Non-conservative substitutions will entail exchanging a member ofone of these classes for another class. Such substituted residues alsomay be introduced into the conservative substitution sites or, morepreferably, into the remaining (non-conserved) sites.

[0580] The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al. Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

[0581] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

[0582] 5.2.2. Modifications of PRO Polypeptides

[0583] Covalent modifications of PRO polypeptides are included withinthe scope of this invention. One type of covalent modification includesreacting targeted amino acid residues of a PRO polypeptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of the PRO polypeptide.Derivatization with bifunctional agents is useful, for instance, forcrosslinking the PRO polypeptide to a water-insoluble support matrix orsurface for use in the method for purifying anti-PRO antibodies, andvice-versa. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

[0584] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains [T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0585] Another type of covalent modification of the PRO polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in the native sequence PROpolypeptide (either by removing the underlying glycosylation site or bydeleting the glycosylation by chemical and/or enzymatic means), and/oradding one or more glycosylation sites that are not present in thenative sequence PRO polypeptide. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

[0586] Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO polypeptide (forO-linked glycosylation sites). The PRO amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the PRO polypeptide at preselected bases suchthat codons are generated that will translate into the desired aminoacids.

[0587] Another means of increasing the number of carbohydrate moietieson the PRO polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0588] Removal of carbohydrate moieties present on the PRO polypeptidemay be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981).

[0589] Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0590] Another type of covalent modification of the PRO polypeptidecomprises linking the PRO polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

[0591] The PRO polypeptide of the present invention may also be modifiedin a way to form a chimeric molecule comprising the PRO polypeptidefused to another, heterologous polypeptide or amino acid sequence.

[0592] In one embodiment, such a chimeric molecule comprises a fusion ofthe PRO polypeptide with a protein transduction domain which targets thePRO polypeptide for delivery to various tissues and more particularlyacross the brain blood barrier, using, for example, the proteintransduction domain of human immunodeficiency virus TAT protein(Schwarze et al., 1999, Science 285: 1569-72).

[0593] In another embodiment, such a chimeric molecule comprises afusion of the PRO polypeptide with a tag polypeptide which provides anepitope to which an anti-tag antibody can selectively bind. The epitopetag is generally placed at the amino- or carboxyl-terminus of the PROpolypeptide. The presence of such epitope-tagged forms of the PROpolypeptide can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables the PROpolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-His) orpoly-histidine-glycine (poly-His-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266: 15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

[0594] In an alternative embodiment, the chimeric molecule may comprisea fusion of the PRO polypeptide with an immunoglobulin or a particularregion of an immunoglobulin. For a bivalent form of the chimericmolecule (also referred to as an“immunoadhesin”), such a fusion could beto the Fc region of an IgG molecule. The Ig fusions preferably includethe substitution of a soluble (transmembrane domain deleted orinactivated) form of a PRO polypeptide in place of at least one variableregion within an Ig molecule. In a particularly preferred embodiment,the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also, U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

[0595] 5.2.3. Preparation of the PRO Polypeptide

[0596] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO polypeptides. In particular, cDNAs encoding PROpolypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below. It is noted that proteins produced inseparate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the PRO DNA as well as all furthernative homologues and variants included in the foregoing definition ofPRO polypeptides, will be referred to as“PRO” regardless of their originor mode of preparation.

[0597] The description below relates primarily to production of PROpolypeptides by culturing cells transformed or transfected with a vectorcontaining nucleic acid encoding PRO polypeptides. It is, of course,contemplated that alternative methods that are well known in the art maybe employed to prepare the PRO polypeptide. For instance, the PROpolypeptide sequence, or portions thereof, may be produced by directpeptide synthesis using solid-phase techniques. See, e.g., Stewart etal., Solid-Phase Peptide Synthesis (W. H. Freeman Co.: San Francisco,Calif., 1969); Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963). Invitro protein synthesis may be performed using manual techniques or byautomation. Automated synthesis maybe accomplished, for instance, withan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of the PRO polypeptide maybe chemically synthesized separately and combined using chemical orenzymatic methods to produce the full-length PRO polypeptide.

[0598] 5.2.3.1. Isolation of DNA Encoding PRO Polypeptides

[0599] DNA encoding the PRO polypeptide may be obtained from a cDNAlibrary prepared from tissue believed to possess the mRNA encoding thePRO polypeptide and to express it at a detectable level. Accordingly,DNAs encoding the human PRO polypeptide can be conveniently obtainedfrom cDNA libraries prepared from human tissues, such as described inthe Examples. The gene encoding the PRO polypeptide may also be obtainedfrom a genomic library or by oligonucleotide synthesis.

[0600] Libraries can be screened with probes (such as antibodies to thePRO polypeptide or oligonucleotides of at least about 20-80 bases)designed to identify the gene of interest or the protein encoded by it.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures, such as described in Sambrook etal., supra. An alternative means to isolate the gene encoding the PROpolypeptide is to use PCR methodology. Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1995).

[0601] The Examples below describe techniques for screening a cDNAlibrary. The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The oligonucleotide is preferably labeled such that it can bedetected upon hybridization to DNA in the library being screened.Methods of labeling are well known in the art, and include the use ofradiolabels like ³²P-labeled ATP, biotinylation, or enzyme labeling.Hybridization conditions, including moderate stringency and highstringency, are provided in Sambrook et al., supra.

[0602] Sequences identified in such library screening methods can becompared and aligned to other known sequences deposited and available inpublic databases such as GenBank or other private sequence databases.Sequence identity (at either the amino acid or nucleotide level) withindefined regions of the molecule or across the full-length sequence canbe determined through sequence alignment using computer softwareprograms such as ALIGN, DNAstar, and INHERIT, which employ variousalgorithms to measure homology.

[0603] Nucleic acid having protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

[0604] 5.2.3.2. Selection and Transformation of Host Cells

[0605] Host cells are transfected or transformed with expression orcloning vectors described herein for PRO polypeptide production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH, and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: A Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0606] Methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ treatment and electroporation. Depending onthe host cell used, transformation is performed using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described in Sambrook et al., supra, orelectroporation is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al , Gene, 23: 315 (1983) and WO 89/05859 publishedJun. 29, 1989. For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham and van der Eb, Virology,52:456-457 (1978) can be employed. General aspects of mammalian cellhost system transformations have been described in U.S. Pat. No.4,399,216. Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130: 946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979).However, other methods for introducing DNA into cells, such as bynuclear microinjection, electroporation, bacterial protoplast fusionwith intact cells, or polycations, e.g., polybrene or polyornithine, mayalso be used. For various techniques for transforming mammalian cells,see, Keown et al., Methods in Enzymology, 185: 527-537 (1990) andMansour et al., Nature, 336: 348-352 (1988).

[0607] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include, but are not limited to, eubacteria, suchas Gram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325); and K5772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B licheniformis(e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

[0608] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forvectors encoding the PRO polypeptide. Saccharomyces cerevisiae is acommonly used lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published May 2, 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9: 968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8: 135(1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,28: 265-278 [1988]); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published Oct. 31, 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published Jan.10, 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112: 284-289 [1983]; Tilbumn et al.,Gene, 26: 205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4: 475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces,

[0609] Torulopsis, and Rhodotorula. A list of specific species that areexemplary of this class of yeasts may be found in C. Anthony, TheBiochemistry of Methylotrophs, 269 (1982).

[0610] Suitable host cells for the expression of nucleic acid encodingglycosylated PRO polypeptides are derived from multicellular organisms.Examples of invertebrate cells include insect cells such as DrosophilaS2 and Spodoptera Sf9, as well as plant cells. Examples of usefulmammalian host cell lines include Chinese hamster ovary (CHO) and COScells. More specific examples include monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. Gen.Virol., 36: 59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlauband Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertolicells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mousemammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriatehost cell is deemed to be within the skill in the art.

[0611] 5.2.3.3. Selection and Use of a Replicable Vector

[0612] The nucleic acid (e.g., cDNA or genomic DNA) encoding the PROpolypeptide may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence if thesequence is to be secreted, an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques that are known tothe skilled artisan.

[0613] The PRO polypeptide may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the DNA encoding the PRO polypeptide thatis inserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published Apr. 4, 1990), or the signal described inWO 90/13646 published Nov. 15, 1990. In mammalian cell expression,mammalian signal sequences may be used to direct secretion of theprotein, such as signal sequences from secreted polypeptides of the sameor related species, as well as viral secretory leaders.

[0614] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV, or BPV) are useful for cloning vectors in mammaliancells.

[0615] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0616] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thenucleic acid encoding the PRO polypeptide such as DHFR or thymidinekinase. An appropriate host cell when wild-type DHFR is employed is theCHO cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980).A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7. Stinchcomb et al., Nature, 282: 39 (1979);Kingsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157(1980). The trp1 gene provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example, ATCC No.44076 or PEP4-1. Jones, Genetics, 85: 12 (1977).

[0617] Expression and cloning vectors usually contain a promoteroperably linked to the nucleic acid sequence encoding the PROpolypeptide to direct mRNA synthesis. Promoters recognized by a varietyof potential host cells are well known. Promoters suitable for use withprokaryotic hosts include the β-lactamase and lactose promoter systems(Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544(1979)), alkaline phosphatase, a tryptophan (trp) promoter system(Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP 36,776), and hybridpromoters such as the tac promoter (deBoer et al., Proc. Natl. Acad.Sci. USA, 80: 21-25 (1983)). Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S. D.) sequence operably linked to theDNA encoding the PRO polypeptide.

[0618] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17: 4900(1978)), such as enolase, glyceralde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0619] Other yeast promoters that are inducible promoters having theadditional advantage of transcription controlled by growth conditionsare the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionem, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0620] PRO nucleic acid transcription from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus, and Simian Virus 40 (SV40); by heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter; and byheat-shock promoters, provided such promoters are compatible with thehost cell systems.

[0621] Transcription of a DNA encoding the PRO polypeptide by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thesequence coding for PRO polypeptides, but is preferably located at asite 5′ from the promoter.

[0622] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the PRO polypeptide.

[0623] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of the PRO polypeptide in recombinantvertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281: 40-46 (1979); EP 117,060;and EP 117,058.

[0624] 5.2.3.4. Detecting Gene Amplification/Expression

[0625] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0626] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native-sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to DNAencoding the PRO polypeptide and encoding a specific antibody epitope.

[0627] 5.2.3.5. Purification of PRO Polypeptides

[0628] Forms of PRO polypeptides may be recovered from culture medium orfrom host cell lysates. If membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g., TRITON-X™ 100) or byenzymatic cleavage. Cells employed in expression of nucleic acidencoding the PRO polypeptide can be disrupted by various physical orchemical means, such as freeze-thaw cycling, sonication, mechanicaldisruption, or cell-lysing agents. It may be desired to purify the PROpolypeptide from recombinant cell proteins or polypeptides. Thefollowing procedures are exemplary of suitable purification procedures:by fractionation on an ion-exchange column; ethanolprecipitation;reverse phase HPLC; chromatography on silica or on a cation-exchangeresin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfateprecipitation; gel filtration using, for example, Sephadex G-75; proteinA Sepharose columns to remove contaminants such as IgG; and metalchelating columns to bind epitope-tagged forms of the PRO polypeptide.Various methods of protein purification may be employed and such methodsare known in the art and described, for example, in Deutscher, Methodsin Enzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice (Springer-Verlag: New York, 1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular PRO polypeptide produced.

[0629] 5.2.4. Uses of PRO Polypeptides

[0630] 5.2.4.1. Assays for Cardiovascular, Endothelial, and AngiogenicActivity

[0631] Various assays can be used to test the polypeptide herein forcardiovascular, endothelial, and angiogenic activity. Such assaysinclude those provided in the Examples below.

[0632] Assays for testing for endothelin antagonist activity, asdisclosed in U.S. Pat. No. 5,773,414, include a rat heart ventriclebinding assay where the polypeptide is tested for its ability to inhibitiodinized endothelin-1 binding in a receptor assay, an endothelinreceptor binding assay testing for intact cell binding of radiolabeledendothelin-1using rabbit renal artery vascular smooth muscle cells, aninositol phosphate accumulation assay where functional activity isdetermined in Rat-1 cells by measuring intra-cellular levels of secondmessengers, an arachidonic acid release assay that measures the abilityof added compounds to reduce endothelin-stimulated arachidonic acidrelease in cultured vascular smooth muscles, in vitro (isolated vessel)studies using endothelium from male New Zealand rabbits, and in vivostudies using male Sprague-Dawley rats.

[0633] Assays for tissue generation activity include, withoutlimitation, those described in WO 95/16035 (bone, cartilage, tendon); WO95/05846 (nerve, neuronal), and WO 91/07491 (skin, endothelium).

[0634] Assays for wound-healing activity include, for example, thosedescribed in Winter, Epidermal Wound Healing, Maibach, H I and Rovee, DT, eds. (Year Book Medical Publishers, Inc., Chicago), pp. 71-112, asmodified by the article of Eaglstein and Mertz, J. Invest. Dermatol.,71: 382-384 (1978).

[0635] An assay to screen for a test molecule relating to a PROpolypeptide that binds an endothelin B₁ (ETB₁) receptor polypeptide andmodulates signal transduction activity involves providing a host celltransformed with a DNA encoding endothelin B₁ receptor polypeptide,exposing the cells to the test candidate, and measuring endothelin B₁receptor signal transduction activity, as described, e.g., in U.S. Pat.No. 5,773,223.

[0636] There are several cardiac hypertrophy assays. In vitro assaysinclude induction of spreading of adult rat cardiac myocytes. In thisassay, ventricular myocytes are isolated from a single (maleSprague-Dawley) rat, essentially following a modification of theprocedure described in detail by Piper et al., “Adult ventricular ratheart muscle cells” in Cell Culture Techniques in Heart and VesselResearch, H. M. Piper, ed. (Berlin: Springer-Verlag, 1990), pp. 36-60.This procedure permits the isolation of adult ventricular myocytes andthe long-term culture of these cells in the rod-shaped phenotype.Phenylephrine and Prostaglandin F_(2α) (PGF_(2α)) have been shown toinduce a spreading response in these adult cells. The inhibition ofmyocyte spreading induced by PGF_(2α) or PGF_(2α) analogs (e g.,fluprostenol) and phenylephrine by various potential inhibitors ofcardiac hypertrophy is then tested.

[0637] One example of an in vivo assay is a test for inhibiting cardiachypertrophy induced by fluprostenol in vivo.

[0638] This pharmacological model tests the ability of the PROpolypeptide to inhibit cardiac hypertrophy induced in rats (e.g., maleWistar or Sprague-Dawley) by subcutaneous injection of fluprostenol (anagonist analog of PGF_(2α)). It is known that rats with pathologiccardiac hypertrophy induced by myocardial infarction have chronicallyelevated levels of extractable PGF_(2α) in their myocardium. Lai et al.,Am. J. Physiol. (Heart Circ. Physiol.), 271: H2197-H2208 (1996).Accordingly, factors that can inhibit the effects of fluprostenol onmyocardial growth in vivo are potentially useful for treating cardiachypertrophy. The effects of the PRO polypeptide on cardiac hypertrophyare determined by measuring the weight of heart, ventricles, and leftventricle (normalized by body weight) relative to fluprostenol-treatedrats not receiving the PRO polypeptide.

[0639] Another example of an in vivo assay is the pressure-overloadcardiac hypertrophy assay. For in vivo testing it is common to inducepressure-overload cardiac hypertrophy by constriction of the abdominalaorta of test animals. In a typical protocol, rats (e.g., male Wistar orSprague-Dawley) are treated under anesthesia, and the abdominal aorta ofeach rat is narrowed down just below the diaphragm. Beznak M., Can. J.Biochem. Physiol., 33: 985-94 (1955). The aorta is exposed through asurgical incision, and a blunted needle is placed next to the vessel.The aorta is constricted with a ligature of silk thread around theneedle, which is immediately removed and which reduces the lumen of theaorta to the diameter of the needle. This approach is described, forexample, in Rossi et al., Am. Heart J., 124: 700-709 (1992) and O'Rourkeand Reibel, P.S.E.M.B., 200: 95-100 (1992).

[0640] In yet another in vivo assay, the effect on cardiac hypertrophyfollowing experimentally induced myocardial infarction (MI) is measured.Acute MI is induced in rats by left coronary artery ligation andconfirmed by electrocardiographic examination. A sham-operated group ofanimals is also prepared as control animals. Earlier data have shownthat cardiac hypertrophy is present in the group of animals with MI, asevidenced by an 18% increase in heart weight-to-body weight ratio. Laiet al., supra. Treatment of these animals with candidate blockers ofcardiac hypertrophy, e.g., the PRO polypeptide, provides valuableinformation about the therapeutic potential of the candidates tested.One further such assay test for induction of cardiac hypertrophy isdisclosed in U.S. Pat. No. 5,773,415, using Sprague-Dawley rats.

[0641] For cancer, a variety of well-known animal models can be used tofurther understand the role of the genes identified herein in thedevelopment and pathogenesis of tumors, and to test the efficacy ofcandidate therapeutic agents, including antibodies and other antagonistsof native PRO polypeptides, such as small-molecule antagonists. The invivo nature of such models makes them particularly predictive ofresponses in human patients. Animal models of tumors and cancers (e.g.,breast cancer, colon cancer, prostate cancer, lung cancer, etc.) includeboth non-recombinant and recombinant (transgenic) animals.Non-recombinant animal models include, for example, rodent, e.g., murinemodels. Such models can be generated by introducing tumor cells intosyngeneic mice using standard techniques, e.g., subcutaneous injection,tail vein injection, spleen implantation, intraperitoneal implantation,implantation under the renal capsule, or orthopin implantation, e.g.,colon cancer cells implanted in colonic tissue. See, e.g., PCTpublication No. WO 97/33551, published Sep. 18, 1997. Probably the mostoften used animal species in oncological studies are immunodeficientmice and, in particular, nude mice. The observation that the nude mousewith thymic hypo/aplasia could successfully act as a host for humantumor xenografts has lead to its widespread use for this purpose. Theautosomal recessive nu gene has been introduced into a very large numberof distinct congenic strains of nude mouse, including, for example, ASW,A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st,NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII, and SJL. In addition, awide variety of other animals with inherited immunological defects otherthan the nude mouse have been bred and used as recipients of tumorxenografts. For further details see, e.g., The Nude Mouse in OncologyResearch, E. Boven and B. Winograd, eds. (CRC Press, Inc., 1991).

[0642] The cells introduced into such animals can be derived from knowntumor/cancer cell lines, such as any of the above-listed tumor celllines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cellline transfected with the neu protooncogene); ras-transfected NIH-3T3cells; Caco-2 (ATCC HTB-37); or a moderately well-differentiated gradeII human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38); or fromtumors and cancers. Samples of tumor or cancer cells can be obtainedfrom patients undergoing surgery, using standard conditions involvingfreezing and storing in liquid nitrogen. Karmali et al., Br. J. Cancer,48: 689-696 (1983).

[0643] Tumor cells can be introduced into animals such as nude mice by avariety of procedures. The subcutaneous (s.c.) space in mice is verysuitable for tumor implantation. Tumors can be transplanted s.c. assolid blocks, as needle biopsies by use of a trochar, or as cellsuspensions. For solid-block or trochar implantation, tumor tissuefragments of suitable size are introduced into the s.c. space. Cellsuspensions are freshly prepared from primary tumors or stable tumorcell lines, and injected subcutaneously. Tumor cells can also beinjected as subdermal implants. In this location, the inoculum isdeposited between the lower part of the dermal connective tissue and thes.c. tissue.

[0644] Animal models of breast cancer can be generated, for example, byimplanting rat neuroblastoma cells (from which the neu oncogene wasinitially isolated), or neu-transformed NIH-3T3 cells into nude mice,essentially as described by Drebin et al. Proc. Nat. Acad. Sci. USA, 83:9129-9133 (1986).

[0645] Similarly, animal models of colon cancer can be generated bypassaging colon cancer cells in animals, e.g., nude mice, leading to theappearance of tumors in these animals. An orthotopic transplant model ofhuman colon cancer in nude mice has been described, for example, by Wanget al, Cancer Research, 54: 4726-4728 (1994) and Too et al., CancerResearch, 55: 681-684 (1995). This model is based on theso-called“METAMOUSE™” sold by AntiCancer, Inc., (San Diego, Calif.).

[0646] Tumors that arise in animals can be removed and cultured invitro. Cells from the in vitro cultures can then be passaged to animals.Such tumors can serve as targets for further testing or drug screening.Alternatively, the tumors resulting from the passage can be isolated andRNA from pre-passage cells and cells isolated after one or more roundsof passage analyzed for differential expression of genes of interest.Such passaging techniques can be performed with any known tumor orcancer cell lines.

[0647] For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 arechemically induced fibrosarcomas of BALB/c female mice (DeLeo et al., J.Exp. Med., 146: 720 (1977)), which provide a highly controllable modelsystem for studying the anti-tumor activities of various agents.Palladino et al., J. Immunol., 138: 4023-4032 (1987). Briefly, tumorcells are propagated in vitro in cell culture. Prior to injection intothe animals, the cell lines are washed and suspended in buffer, at acell density of about 10×10⁶ to 10×10⁷ cells/ml. The animals are theninfected subcutaneously with 10 to 100 μl of the cell suspension,allowing one to three weeks for a tumor to appear.

[0648] In addition, the Lewis lung (3LL) carcinoma of mice, which is oneof the most thoroughly studied experimental tumors, can be used as aninvestigational tumor model. Efficacy in this tumor model has beencorrelated with beneficial effects in the treatment of human patientsdiagnosed with small-cell carcinoma of the lung (SCCL). This tumor canbe introduced in normal mice upon injection of tumor fragments from anaffected mouse or of cells maintained in culture. Zupi et al., Br. J.Cancer, 41: suppl. 4, 30 (1980). Evidence indicates that tumors can bestarted from injection of even a single cell and that a very highproportion of infected tumor cells survive. For further informationabout this tumor model see, Zacharski, Haemostasis, 16: 300-320 (1986).

[0649] One way of evaluating the efficacy of a test compound in ananimal model with an implanted tumor is to measure the size of the tumorbefore and after treatment. Traditionally, the size of implanted tumorshas been measured with a slide caliper in two or three dimensions. Themeasure limited to two dimensions does not accurately reflect the sizeof the tumor; therefore, it is usually converted into the correspondingvolume by using a mathematical formula. However, the measurement oftumor size is very inaccurate. The therapeutic effects of a drugcandidate can be better described as treatment-induced growth delay andspecific growth delay. Another important variable in the description oftumor growth is the tumor volume doubling time. Computer programs forthe calculation and description of tumor growth are also available, suchas the program reported by Rygaard and Spang-Thomsen, Proc. 6th Int.Workshop on Immune-Deficient Animals, Wu and Sheng eds. (Basel, 1989),p. 301. It is noted, however, that necrosis and inflammatory responsesfollowing treatment may actually result in an increase in tumor size, atleast initially. Therefore, these changes need to be carefullymonitored, by a combination of a morphometric method and flow cytometricanalysis.

[0650] Further, recombinant (transgenic) animal models can be engineeredby introducing the coding portion of the PRO gene identified herein intothe genome of animals of interest, using standard techniques forproducing transgenic animals. Animals that can serve as a target fortransgenic manipulation include, without limitation, mice, rats,rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g.,baboons, chimpanzees and monkeys. Techniques known in the art tointroduce a transgene into such animals include pronucleicmicroinjection (U.S. Pat. No. 4,873,191); retrovirus-mediated genetransfer into germ lines (e.g., Van der Putten et al., Proc. Natl. Acad.Sci. USA, 82: 6148-615 (1985)); gene targeting in embryonic stem cells(Thompson et al., Cell, 56: 313-321 (1989)); electroporation of embryos(Lo, Mol. Cell. Biol., 3: 1803-1814 (1983)); and sperm-mediated genetransfer. Lavitrano et al., Cell, 57: 717-73 (1989). For a review, seefor example, U.S. Pat. No. 4,736,866.

[0651] For the purpose of the present invention, transgenic animalsinclude those that carry the transgene only in part of their cells(“mosaic animals”). The transgene can be integrated either as a singletransgene, or in concatamers, e.g., head-to-head or head-to-tailtandems. Selective introduction of a transgene into a particular celltype is also possible by following, for example, the technique of Laskoet al., Proc. Natl. Acad. Sci. USA, 89: 6232-636 (1992). The expressionof the transgene in transgenic animals can be monitored by standardtechniques. For example, Southern blot analysis or PCR amplification canbe used to verify the integration of the transgene. The level of mRNAexpression can then be analyzed using techniques such as in situhybridization, Northern blot analysis, PCR, or immunocytochemistry. Theanimals are further examined for signs of tumor or cancer development.

[0652] Alternatively, “knock-out” animals can be constructed that have adefective or altered gene encoding a PRO polypeptide identified herein,as a result of homologous recombination between the endogenous geneencoding the PRO polypeptide and altered genomic DNA encoding the samepolypeptide introduced into an embryonic cell of the animal. Forexample, cDNA encoding a particular PRO polypeptide can be used to clonegenomic DNA encoding that polypeptide in accordance with establishedtechniques. A portion of the genomic DNA encoding a particular PROpolypeptide can be deleted or replaced with another gene, such as a geneencoding a selectable marker that can be used to monitor integration.Typically, several kilobases of unaltered flanking DNA (both at the 5′and 3′ ends) are included in the vector. See, e.g., Thomas and Capecchi,Cell, 51: 503 (1987) for a description of homologous recombinationvectors. The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected. See, e.g.,Li et al., Cell, 69: 915 (1992). The selected cells are then injectedinto a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras. See, e g , Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL:Oxford, 1987), pp. 113-152. A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm to create a “knock-out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized, for instance, by their ability to defend against certainpathological conditions and by their development of pathologicalconditions due to absence of the PRO polypeptide.

[0653] The efficacy of antibodies specifically binding the PROpolypeptides identified herein, and other drug candidates, can be testedalso in the treatment of spontaneous animal tumors. A suitable targetfor such studies is the feline oral squamous cell carcinoma (SCC).Feline oral SCC is a highly invasive, malignant tumor that is the mostcommon oral malignancy of cats, accounting for over 60% of the oraltumors reported in this species. It rarely metastasizes to distantsites, although this low incidence of metastasis may merely be areflection of the short survival times for cats with this tumor. Thesetumors are usually not amenable to surgery, primarily because of theanatomy of the feline oral cavity. At present, there is no effectivetreatment for this tumor. Prior to entry into the study, each catundergoes complete clinical examination and biopsy, and is scanned bycomputed tomography (CT). Cats diagnosed with sublingual oral squamouscell tumors are excluded from the study. The tongue can become paralyzedas a result of such tumor, and even if the treatment kills the tumor,the animals may not be able to feed themselves. Each cat is treatedrepeatedly, over a longer period of time. Photographs of the tumors willbe taken daily during the treatment period, and at each subsequentrecheck. After treatment, each cat undergoes another CT scan. CT scansand thoracic radiograms are evaluated every 8 weeks thereafter. The dataare evaluated for differences in survival, response, and toxicity ascompared to control groups. Positive response may require evidence oftumor regression, preferably with improvement of quality of life and/orincreased life span.

[0654] In addition, other spontaneous animal tumors, such asfibrosarcoma, adenocarcinoma, lymphoma, chondroma, or leiomyosarcoma ofdogs, cats, and baboons can also be tested. Of these, mammaryadenocarcinoma in dogs and cats is a preferred model as its appearanceand behavior are very similar to those in humans. However, the use ofthis model is limited by the rare occurrence of this type of tumor inanimals.

[0655] Other in vitro and in vivo cardiovascular, endothelial, andangiogenic tests known in the art are also suitable herein.

[0656] 5.2.4.2. Tissue Distribution

[0657] The results of the cardiovascular, endothelial, and angiogenicassays herein can be verified by further studies, such as by determiningmRNA expression in various human tissues.

[0658] As noted before, gene amplification and/or gene expression invarious tissues may be measured by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA (Thomas, Proc.Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis),or in situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.

[0659] Gene expression in various tissues, alternatively, may bemeasured by immunological methods, such as immunohistochemical stainingof tissue sections and assay of cell culture or body fluids, toquantitate directly the expression of gene product. Antibodies usefulfor immunohistochemical staining and/or assay of sample fluids may beeither monoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native-sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope. General techniques forgenerating antibodies, and special protocols for in situ hybridizationare provided hereinbelow.

[0660] 5.2.4.3. Antibody Binding Studies

[0661] The results of the cardiovascular, endothelial, and angiogenicstudy can be further verified by antibody binding studies, in which theability of anti-PRO antibodies to inhibit the effect of the PROpolypeptides on endothelial cells or other cells used in thecardiovascular, endothelial, and angiogenic assays is tested. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies, the preparation of which will be describedhereinbelow.

[0662] Antibody binding studies may be carried out in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: AManual of Techniques (CRC Press, Inc., 1987), pp. 147-158.

[0663] Competitive binding assays rely on the ability of a labeledstandard to compete with the test sample analyte for binding with alimited amount of antibody. The amount of target protein in the testsample is inversely proportional to the amount of standard that becomesbound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies preferably are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte that remain unbound.

[0664] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody that is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

[0665] For immunohistochemistry, the tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin, for example.

[0666] 5.2.4.4. Cell-based Tumor Assays

[0667] Cell-based assays and animal models for cardiovascular,endothelial, and angiogenic disorders, such as tumors, can be used toverify the findings of a cardiovascular, endothelial, and angiogenicassay herein, and further to understand the relationship between thegenes identified herein and the development and pathogenesis ofundesirable cardiovascular, endothelial, and angiogenic cell growth. Therole of gene products identified herein in the development and pathologyof undesirable cardiovascular, endothelial, and angiogenic cell growth,e.g., tumor cells, can be tested by using cells or cells lines that havebeen identified as being stimulated or inhibited by the PRO polypeptideherein. Such cells include, for example, those set forth in the Examplesbelow.

[0668] In a different approach, cells of a cell type known to beinvolved in a particular cardiovascular, endothelial, and angiogenicdisorder are transfected with the cDNAs herein, and the ability of thesecDNAs to induce excessive growth or inhibit growth is analyzed. If thecardiovascular, endothelial, and angiogenic disorder is cancer, suitabletumor cells include, for example, stable tumor cell lines such as theB104-1-1 cell line (stable NIH-3T3 cell line transfected with the neuprotooncogene) and ras-transfected NIH-3T3 cells, which can betransfected with the desired gene and monitored for tumorigenic growth.Such transfected cell lines can then be used to test the ability ofpoly- or monoclonal antibodies or antibody compositions to inhibittumorigenic cell growth by exerting cytostatic or cytotoxic activity onthe growth of the transformed cells, or by mediating antibody-dependentcellular cytotoxicity (ADCC). Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of cardiovascular, endothelial, andangiogenic disorders such as cancer.

[0669] In addition, primary cultures derived from tumors in transgenicanimals (as described above) can be used in the cell-based assaysherein, although stable cell lines are preferred. Techniques to derivecontinuous cell lines from transgenic animals are well known in the art.See, e.g., Small et al., Mol. Cell. Biol., 5: 642-648 (1985).

[0670] 5.2.4.5. Gene Therapy

[0671] Described below are methods and compositions whereby diseasesymptoms may be ameliorated. Certain diseases are brought about, atleast in part, by an excessive level of gene product, or by the presenceof a gene product exhibiting an abnormal or excessive activity. As such,the reduction in the level and/or activity of such gene products wouldbring about the amelioration of such disease symptoms.

[0672] Alternatively, certain other diseases are brought about, at leastin part, by the absence or reduction of the level of gene expression, ora reduction in the level of a gene product's activity. As such, anincrease in the level of gene expression and/or the activity of suchgene products would bring about the amelioration of such diseasesymptoms.

[0673] In some cases, the up-regulation of a gene in a disease statereflects a protective role for that gene product in responding to thedisease condition. Enhancement of such a target gene's expression, orthe activity of the target gene product, will reinforce the protectiveeffect it exerts. Some disease states may result from an abnormally lowlevel of activity of such a protective gene. In these cases also, anincrease in the level of gene expression and/or the activity of suchgene products would bring about the amelioration of such diseasesymptoms.

[0674] The PRO polypeptides described herein and polypeptidyl agonistsand antagonists may be employed in accordance with the present inventionby expression of such polypeptides in vivo, which is often referred toas gene therapy.

[0675] There are two major approaches to getting the nucleic acid(optionally contained in a vector) into the patient's cells: in vivo andex vivo. For in vivo delivery the nucleic acid is injected directly intothe patient, usually at the sites where the PRO polypeptide is required,i.e., the site of synthesis of the PRO polypeptide, if known, and thesite (e.g., wound) where biological activity of the PRO polypeptide isneeded. For ex vivo treatment, the patient's cells are removed, thenucleic acid is introduced into these isolated cells, and the modifiedcells are administered to the patient either directly or, for example,encapsulated within porous membranes that are implanted into the patient(see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a varietyof techniques available for introducing nucleic acids into viable cells.The techniques vary depending upon whether the nucleic acid istransferred into cultured cells in vitro, or transferred in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, transduction, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc.Transduction involves the association of a replication-defective,recombinant viral (preferably retroviral) particle with a cellularreceptor, followed by introduction of the nucleic acids contained by theparticle into the cell. A commonly used vector for ex vivo delivery ofthe gene is a retrovirus.

[0676] The currently preferred in vivo nucleic acid transfer techniquesinclude transfection with viral or non-viral vectors (such asadenovirus, lentivirus, Herpes simplex I virus, or adeno-associatedvirus (AAV)) and lipid-based systems (useful lipids for lipid-mediatedtransfer of the gene are, for example, DOTMA, DOPE, and DC-Chol; see,e.g., Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)). Themost preferred vectors for use in gene therapy are viruses, mostpreferably adenoviruses, AAV, lentiviruses, or retroviruses. A viralvector such as a retroviral vector includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger. Inaddition, a viral vector such as a retroviral vector includes a nucleicacid molecule that, when transcribed in the presence of a gene encodingthe PRO polypeptide, is operably linked thereto and acts as atranslation initiation sequence. Such vector constructs also include apackaging signal, long terminal repeats (LTRs) or portions thereof, andpositive and negative strand primer binding sites appropriate to thevirus used (if these are not already present in the viral vector). Inaddition, such vector typically includes a signal sequence for secretionof the PRO polypeptide from a host cell in which it is placed.Preferably the signal sequence for this purpose is a mammalian signalsequence, most preferably the native signal sequence for the PROpolypeptide. Optionally, the vector construct may also include a signalthat directs polyadenylation, as well as one or more restriction sitesand a translation termination sequence. By way of example, such vectorswill typically include a 5′ LTR, a tRNA binding site, a packagingsignal, an origin of second-strand DNA synthesis, and a 3′ LTR or aportion thereof. Other vectors can be used that are non-viral, such ascationic lipids, polylysine, and dendrimers.

[0677] In some situations, it is desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell-surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins that bind to a cell-surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins that undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262: 4429-4432(1987); and Wagner et al, Proc. Natl.Acad. Sci. USA, 87: 3410-3414 (1990). For a review of the currentlyknown gene marking and gene therapy protocols, see, Anderson et al.,Science, 256: 808-813 (1992). See also WO 93/25673 and the referencescited therein.

[0678] Suitable gene therapy and methods for making retroviral particlesand structural proteins can be found in, e.g., U.S. Pat. No. 5,681,746.

[0679] 5.2.4.6. Use of Gene as a Diagnostic

[0680] This invention is also related to the use of the gene encodingthe PRO polypeptide as a diagnostic. Detection of a mutated form of thePRO polypeptide will allow a diagnosis of a cardiovascular, endothelial,and angiogenic disease or a susceptibility to a cardiovascular,endothelial, and angiogenic disease, such as a tumor, since mutations inthe PRO polypeptide may cause tumors.

[0681] Individuals carrying mutations in the genes encoding a human PROpolypeptide may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells, suchas from blood, urine, saliva, tissue biopsy, and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324: 163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding thePRO polypeptide can be used to identify and analyze the PRO polypeptidemutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled RNA encoding the PRO polypeptide, or alternatively,radiolabeled antisense DNA sequences encoding the PRO polypeptide.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

[0682] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamidine gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures. See, e.g., Myerset al., Science 230: 1242 (1985).

[0683] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method, for example, Cotton et al., Proc. Natl. Acad.Sci. USA, 85: 4397-4401 (1985).

[0684] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing, or the use of restriction enzymes, e.g.,restriction fragment length polymorphisms (RFLP), and Southern blottingof genomic DNA.

[0685] 5.2.4.7. Use to Detect PRO Polypeptide Levels

[0686] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0687] Expression of nucleic acid encoding the PRO polypeptide may belinked to vascular disease or neovascularization associated with tumorformation. If the PRO polypeptide has a signal sequence and the mRNA ishighly expressed in endothelial cells and to a lesser extent in smoothmuscle cells, this indicates that the PRO polypeptide is present inserum. Accordingly, an anti-PRO polypeptide antibody could be used todiagnose vascular disease or neovascularization associated with tumorformation, since an altered level of this PRO polypeptide may beindicative of such disorders.

[0688] A competition assay may be employed wherein antibodies specificto the PRO polypeptide are attached to a solid support and the labeledPRO polypeptide and a sample derived from the host are passed over thesolid support and the amount of label detected attached to the solidsupport can be correlated to a quantity of the PRO polypeptide in thesample.

[0689] 5.2.4.8. Chromosome Mapping

[0690] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0691] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis for the3′-untranslated region is used to rapidly select primers that do notspan more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0692] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes, andpreselection by hybridization to construct chromosome-specific cDNAlibraries.

[0693] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 500 or 600 bases; however, clones larger than 2,000 bp havea higher likelihood of binding to a unique chromosomal location withsufficient signal intensity for simple detection. FISH requires use ofthe clones from which the gene encoding the PRO polypeptide was derived,and the longer the better. For example, 2,000 bp is good, 4,000 bp isbetter, and more than 4,000 is probably not necessary to get goodresults a reasonable percentage of the time. For a review of thistechnique, see, Verma et al., Human Chromosomes: a Manual of BasicTechniques (Pergamon Press, New York, 1988).

[0694] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available online throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion is then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0695] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0696] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0697] 5.2.4.9. Screening Assays for Drug Candidates

[0698] This invention encompasses methods of screening compounds toidentify those that mimic the PRO polypeptide (agonists) or prevent theeffect of the PRO polypeptide (antagonists). Screening assays forantagonist drug candidates are designed to identify compounds that bindor complex with the PRO polypeptide encoded by the genes identifiedherein, or otherwise interfere with the interaction of the encodedpolypeptides with other cellular proteins. Such screening assays willinclude assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates.

[0699] The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

[0700] All assays for antagonists are common in that they call forcontacting the drug candidate with a PRO polypeptide encoded by anucleic acid identified herein under conditions and for a timesufficient to allow these two components to interact.

[0701] In binding assays, the interaction is binding and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, the PRO polypeptide encoded by the geneidentified herein or the drug candidate is immobilized on a solid phase,e.g., on a microtiter plate, by covalent or non-covalent attachments.

[0702] Non-covalent attachment generally is accomplished by coating thesolid surface with a solution of the PRO polypeptide and drying.Alternatively, an immobilized antibody, e.g., a monoclonal antibody,specific for the PRO polypeptide to be immobilized can be used to anchorit to a solid surface. The assay is performed by adding thenon-immobilized component, which may be labeled by a detectable label,to the immobilized component, e.g., the coated surface containing theanchored component. When the reaction is complete, the non-reactedcomponents are removed, e.g., by washing, and complexes anchored on thesolid surface are detected. When the originally non-immobilizedcomponent carries a detectable label, the detection of label immobilizedon the surface indicates that complexing occurred. Where the originallynon-immobilized component does not carry a label, complexing can bedetected, for example, by using a labeled antibody specifically bindingthe immobilized complex.

[0703] If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340: 245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the“two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

[0704] Compounds that interfere with the interaction of a gene encodinga PRO polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

[0705] If the PRO polypeptide has the ability to stimulate theproliferation of endothelial cells in the presence of the co-mitogenConA, then one example of a screening method takes advantage of thisability. Specifically, in the proliferation assay, human umbilical veinendothelial cells are obtained and cultured in 96-well flat-bottomedculture plates (Costar, Cambridge, Mass.) and supplemented with areaction mixture appropriate for facilitating proliferation of thecells, the mixture containing Con-A (Calbiochem, La Jolla, Calif.).Con-A and the compound to be screened are added and after incubation at37° C., cultures are pulsed with ³⁻H-thymidine and harvested onto glassfiber filters (phD; Cambridge Technology, Watertown, Mass.). Mean³⁻H-thymidine incorporation (cpm) of triplicate cultures is determinedusing a liquid scintillation counter (Beckman Instruments, Irvine,Calif.). Significant ³⁻(H)-thymidine incorporation indicates stimulationof endothelial cell proliferation.

[0706] To assay for antagonists, the assay described above is performed;however, in this assay the PRO polypeptide is added along with thecompound to be screened and the ability of the compound to inhibit³⁻(H)thymidine incorporation in the presence of the PRO polypeptideindicates that the compound is an antagonist to the PRO polypeptide.Alternatively, antagonists may be detected by combining the PROpolypeptide and a potential antagonist with membrane-bound PROpolypeptide receptors or recombinant receptors under appropriateconditions for a competitive inhibition assay. The PRO polypeptide canbe labeled, such as by radioactivity, such that the number of PROpolypeptide molecules bound to the receptor can be used to determine theeffectiveness of the potential antagonist. The gene encoding thereceptor can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting. Coligan etal., Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the PRO polypeptide and a cDNA library createdfrom this RNA is divided into pools and used to transfect COS cells orother cells that are not responsive to the PRO polypeptide. Transfectedcells that are grown on glass slides are exposed to the labeled PROpolypeptide. The PRO polypeptide can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to autoradiographic analysis. Positive pools areidentified and sub-pools are prepared and re-transfected using aninteractive sub-pooling and re-screening process, eventually yielding asingle clone that encodes the putative receptor.

[0707] As an alternative approach for receptor identification, thelabeled PRO polypeptide can be photoaffinity-linked with cell membraneor extract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE and exposed to X-ray film. The labeledcomplex containing the receptor can be excised, resolved into peptidefragments, and subjected to protein micro-sequencing. The amino acidsequence obtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

[0708] In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with the labeledPRO polypeptide in the presence of the candidate compound. The abilityof the compound to enhance or block this interaction could then bemeasured.

[0709] The compositions useful in the treatment of cardiovascular,endothelial, and angiogenic disorders include, without limitation,antibodies, small organic and inorganic molecules, peptides,phosphopeptides, antisense and ribozyme molecules, triple-helixmolecules, etc., that inhibit the expression and/or activity of thetarget gene product.

[0710] More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with a PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

[0711] Another potential PRO polypeptide antagonist is an antisense RNAor DNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see, Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. A sequence“complementary” to aportion of an RNA, as referred to herein, means a sequence havingsufficient complementarity to be able to hybridize with the RNA, forminga stable duplex; in the case of double-stranded antisense nucleic acids,a single strand of the duplex DNA may thus be tested, or triplex helixformation may be assayed. The ability to hybridize will depend on boththe degree of complementarity and the length of the antisense nucleicacid. Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the PRO polypeptide (antisense—Okano, Neurochem.,56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression (CRC Press: Boca Raton, Fla., 1988).

[0712] The antisense oligonucleotides can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g, Letsinger, et al., 1989, Proc. Natl. Acad. Sci.U.S.A 86:6553-6556; Lemaitre, et al., 1987, Proc. Natl. Acad. Sci. USA.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents (see,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

[0713] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyammomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid(v),2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and2,6-diaminopurine.

[0714] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0715] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0716] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier, et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue, et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue, et al., 1987, FEBSLett. 215:327-330).

[0717] Oligonucleotides of the invention may be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein, et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin, et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0718] The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of the PRO polypeptide. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation-initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

[0719] Antisense RNA or DNA molecules are generally at least about 5bases in length, about 10 bases in length, about 15 bases in length,about 20 bases in length, about 25 bases in length, about 30 bases inlength, about 35 in length, about 40 bases in length, about 45 bases inlength, about 50 bases in length, about 55 bases in length, about 60bases in length, about 65 bases in length, about 70 bases in length,about 75 bases in length, about 80 in length, about 85 bases in length,about 90 bases in length, about 95 bases in length, about 100 bases inlength, or more.

[0720] Potential antagonists further include small molecules that bindto the active site, the receptor binding site, or growth factor or otherrelevant binding site of the PRO polypeptide, thereby blocking thenormal biological activity of the PRO polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

[0721] Additional potential antagonists are ribozymes, which areenzymatic RNA molecules capable of catalyzing the specific cleavage ofRNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4: 469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

[0722] While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy target gene mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions which form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Myers, 1995, Molecular Biology andBiotechnology: A Comprehensive Desk Reference, VCH Publishers, New York,(see especially FIG. 4, page 833) and in Haseloff and Gerlach, 1988,Nature, 334:585-591, which is incorporated herein by reference in itsentirety.

[0723] Preferably the ribozyme is engineered so that the cleavagerecognition site is located near the 5′ end of the target gene mRNA,i.e., to increase efficiency and minimize the intracellular accumulationof non-functional mRNA transcripts.

[0724] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter“Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site that hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes that targeteight base-pair active site sequences that are present in the targetgene.

[0725] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g., for improved stability, targeting,etc.) and should be delivered to cells that express the target gene invivo. A preferred method of delivery involves using a DNA construct“encoding” the ribozyme under the control of a strong constitutive polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous target genemessages and inhibit translation. Because ribozymes, unlike antisensemolecules, are catalytic, a lower intracellular concentration isrequired for efficiency.

[0726] Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

[0727] These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

[0728] 5.2.4.10. Types of Cardiovascular, Endothelial, and AngiogenicDisorders to be Treated

[0729] The PRO polypeptides, or agonists or antagonists thereto, thathave activity in the cardiovascular, angiogenic, and endothelial assaysdescribed herein, and/or whose gene product has been found to belocalized to the cardiovascular system, are likely to have therapeuticuses in a variety of cardiovascular, endothelial, and angiogenicdisorders, including systemic disorders that affect vessels, such asdiabetes mellitus. Their therapeutic utility could include diseases ofthe arteries, capillaries, veins, and/or lymphatics. Examples oftreatments hereunder include treating muscle wasting disease, treatingosteoporosis, aiding in implant fixation to stimulate the growth ofcells around the implant and therefore facilitate its attachment to itsintended site, increasing IGF stability in tissues or in serum, ifapplicable, and increasing binding to the IGF receptor (since IGF hasbeen shown in vitro to enhance human marrow erythroid and granulocyticprogenitor cell growth).

[0730] The PRO polypeptides or agonists or antagonists thereto may alsobe employed to stimulate erythropoiesis or granulopoiesis, to stimulatewound healing or tissue regeneration and associated therapies concernedwith re-growth of tissue, such as connective tissue, skin, bone,cartilage, muscle, lung, or kidney, to promote angiogenesis, tostimulate or inhibit migration of endothelial cells, and to proliferatethe growth of vascular smooth muscle and endothelial cell production.The increase in angiogenesis mediated by the PRO polypeptide or agonistwould be beneficial to ischemic tissues and to collateral coronarydevelopment in the heart subsequent to coronary stenosis. Antagonistsare used to inhibit the action of such polypeptides, for example, tolimit the production of excess connective tissue during wound healing orpulmonary fibrosis if the PRO polypeptide promotes such production. Thiswould include treatment of acute myocardial infarction and heartfailure.

[0731] Moreover, the present invention provides the treatment of cardiachypertrophy, regardless of the underlying cause, by administering atherapeutically effective dose of the PRO polypeptide, or agonist orantagonist thereto. If the objective is the treatment of human patients,the PRO polypeptide preferably is recombinant human PRO polypeptide(rhPRO polypeptide). The treatment for cardiac hypertrophy can beperformed at any of its various stages, which may result from a varietyof diverse pathologic conditions, including myocardial infarction,hypertension, hypertrophic cardiomyopathy, and valvular regurgitation.The treatment extends to all stages of the progression of cardiachypertrophy, with or without structural damage of the heart muscle,regardless of the underlying cardiac disorder.

[0732] The decision of whether to use the molecule itself or an agonistthereof for any particular indication, as opposed to an antagonist tothe molecule, would depend mainly on whether the molecule hereinpromotes cardiovascularization, genesis of endothelial cells, orangiogenesis or inhibits these conditions. For example, if the moleculepromotes angiogenesis, an antagonist thereof would be useful fortreatment of disorders where it is desired to limit or preventangiogenesis. Examples of such disorders include vascular tumors such ashaemangioma, tumor angiogenesis, neovascularization in the retina,choroid, or cornea, associated with diabetic retinopathy or prematureinfant retinopathy or macular degeneration and proliferativevitreoretinopathy, rheumatoid arthritis, Crolm's disease,atherosclerosis, ovarian hyperstimulation, psoriasis, endometriosisassociated with neovascularization, restenosis subsequent to balloonangioplasty, scar tissue overproduction, for example, that seen in akeloid that forms after surgery, fibrosis after myocardial infarction,or fibrotic lesions associated with pulmonary fibrosis.

[0733] If, however, the molecule inhibits angiogenesis, it would beexpected to be used directly for treatment of the above conditions.

[0734] On the other hand, if the molecule stimulates angiogenesis itwould be used itself (or an agonist thereof) for indications whereangiogenesis is desired such as peripheral vascular disease,hypertension, inflammatory vasculitides, Reynaud's disease and Reynaud'sphenomenon, aneurysms, arterial restenosis, thrombophlebitis,lymphangitis, lymphedema, wound healing and tissue repair, ischemiareperfusion injury, angina, myocardial infarctions such as acutemyocardial infarctions, chronic heart conditions, heart failure such ascongestive heart failure, and osteoporosis.

[0735] If, however, the molecule inhibits angiogenesis, an antagonistthereof would be used for treatment of those conditions whereangiogenesis is desired.

[0736] Specific types of diseases are described below, where the PROpolypeptide herein or agonists or antagonists thereof may serve asuseful for vascular-related drug targeting or as therapeutic targets forthe treatment or prevention of the disorders. Atherosclerosis is adisease characterized by accumulation of plaques of intimal thickeningin arteries, due to accumulation of lipids, proliferation of smoothmuscle cells, and formation of fibrous tissue within the arterial wall.The disease can affect large, medium, and small arteries in any organ.Changes in endothelial and vascular smooth muscle cell function areknown to play an important role in modulating the accumulation andregression of these plaques.

[0737] Hypertension is characterized by raised vascular pressure in thesystemic arterial, pulmonary arterial, or portal venous systems.Elevated pressure may result from or result in impaired endothelialfunction and/or vascular disease

[0738] Inflammatory vasculitides include giant cell arteritis,Takayasu's arteritis, polyarteritis nodosa (including themicroangiopathic form), Kawasaki's disease, microscopic polyangiitis,Wegener's granulomatosis, and a variety of infectious-related vasculardisorders (including Henoch-Schonlein prupura). Altered endothelial cellfunction has been shown to be important in these diseases.

[0739] Reynaud's disease and Reynaud's phenomenon are characterized byintermittent abnormal impairment of the circulation through theextremities on exposure to cold. Altered endothelial cell function hasbeen shown to be important in this disease.

[0740] Aneurysms are saccular or fusiform dilatations of the arterial orvenous tree that are associated with altered endothelial cell and/orvascular smooth muscle cells.

[0741] Arterial restenosis (restenosis of the arterial wall) may occurfollowing angioplasty as a result of alteration in the function andproliferation of endothelial and vascular smooth muscle cells.

[0742] Thrombophlebitis and lymphangitis are inflammatory disorders ofveins and lymphatics, respectively, that may result from, and/or in,altered endothelial cell function. Similarly, lymphedema is a conditioninvolving impaired lymphatic vessels resulting from endothelial cellfunction.

[0743] The family of benign and malignant vascular tumors arecharacterized by abnormal proliferation and growth of cellular elementsof the vascular system. For example, lymphangiomas are benign tumors ofthe lymphatic system that are congenital, often cystic, malformations ofthe lymphatics that usually occur in newborns. Cystic tumors tend togrow into the adjacent tissue. Cystic tumors usually occur in thecervical and axillary region. They can also occur in the soft tissue ofthe extremities. The main symptoms are dilated, sometimes reticular,structured lymphatics and lymphocysts surrounded by connective tissue.Lymphangiomas are assumed to be caused by improperly connected embryoniclymphatics or their deficiency. The result is impaired local lymphdrainage. Griener et al., Lymphology, 4: 140-144 (1971).

[0744] Another use for the PRO polypeptides herein or agonists orantagonists thereto is in the prevention of tumor angiogenesis, whichinvolves vascularization of a tumor to enable it to growth and/ormetastasize. This process is dependent on the growth of new bloodvessels. Examples of neoplasms and related conditions that involve tumorangiogenesis include breast carcinomas, lung carcinomas, gastriccarcinomas, esophageal carcinomas, colorectal carcinomas, livercarcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervicalcarcinomas, endometrial carcinoma, endometrial hyperplasia,endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer,nasopharyngeal carcinoma, laryngeal carcinomas, bepatoblastoma, Kaposi'ssarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma,glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma,neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas,urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cellcarcinoma, prostate carcinoma, abnormal vascular proliferationassociated with phakomatoses, edema (such as that associated with braintumors), and Meigs' syndrome.

[0745] Age-related macular degeneration (AMD) is a leading cause ofsevere visual loss in the elderly population. The exudative form of AMDis characterized by choroidal neovascularization and retinal pigmentepithelial cell detachment. Because choroidal neovascularization isassociated with a dramatic worsening in prognosis, the PRO polypeptideor agonist or antagonist thereto is expected to be useful in reducingthe severity of AMD.

[0746] Healing of trauma such as wound healing and tissue repair is alsoa targeted use for the PRO polypeptides herein or their agonists orantagonists. Formation and regression of new blood vessels is essentialfor tissue healing and repair. This category includes bone, cartilage,tendon, ligament, and/or nerve tissue growth or regeneration, as well aswound healing and tissue repair and replacement, and in the treatment ofburns, incisions, and ulcers. A PRO polypeptide or agonist or antagonistthereof that induces cartilage and/or bone growth in circumstances wherebone is not normally formed has application in the healing of bonefractures and cartilage damage or defects in humans and other animals.Such a preparation employing a PRO polypeptide or agonist or antagonistthereof may have prophylactic use in closed as well as open fracturereduction and also in the improved fixation of artificial joints. Denovo bone formation induced by an osteogenic agent contributes to therepair of congenital, trauma-induced, or oncologic, resection-inducedcraniofacial defects, and also is useful in cosmetic plastic surgery.

[0747] PRO polypeptides or agonists or antagonists thereto may also beuseful to promote better or faster closure of non-healing wounds,including without limitation pressure ulcers, ulcers associated withvascular insufficiency, surgical and traumatic wounds, and the like.

[0748] It is expected that a PRO polypeptide or agonist or antagonistthereto may also exhibit activity for generation or regeneration ofother tissues, such as organs (including, for example, pancreas, liver,intestine, kidney, skin, or endothelium), muscle (smooth, skeletal, orcardiac), and vascular (including vascular endothelium) tissue, or forpromoting the growth of cells comprising such tissues. Part of thedesired effects may be by inhibition or modulation of fibrotic scarringto allow normal tissue to regenerate.

[0749] A PRO polypeptide herein or agonist or antagonist thereto mayalso be useful for gut protection or regeneration and treatment of lungor liver fibrosis, reperfusion injury in various tissues, and conditionsresulting from systemic cytokine damage. Also, the PRO polypeptide oragonist or antagonist thereto may be useful for promoting or inhibitingdifferentiation of tissues described above from precursor tissues orcells, or for inhibiting the growth of tissues described above.

[0750] A PRO polypeptide or agonist or antagonist thereto may also beused in the treatment of periodontal diseases and in other tooth-repairprocesses. Such agents may provide an environment to attractbone-forming cells, stimulate growth of bone-forming cells, or inducedifferentiation of progenitors of bone-forming cells. A PRO polypeptideherein or an agonist or an antagonist thereto may also be useful in thetreatment of osteoporosis or osteoarthritis, such as through stimulationof bone and/or cartilage repair or by blocking inflammation or processesof tissue destruction (collagenase activity, osteoclast activity, etc.)mediated by inflammatory processes, since blood vessels play animportant role in the regulation of bone turnover and growth.

[0751] Another category of tissue regeneration activity that may beattributable to the PRO polypeptide herein or agonist or antagonistthereto is tendon/ligament formation. A protein that inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed has application in the healingof tendon or ligament tears, deformities, and other tendon or ligamentdefects in humans and other animals. Such a preparation may haveprophylactic use in preventing damage to tendon or ligament tissue, aswell as use in the improved fixation of tendon or ligament to bone orother tissues, and in repairing defects to tendon or ligament tissue. Denovo tendon/ligament-like tissue formation induced by a composition ofthe PRO polypeptide herein or agonist or antagonist thereto contributesto the repair of congenital, trauma-induced, or other tendon or ligamentdefects of other origin, and is also useful in cosmetic plastic surgeryfor attachment or repair of tendons or ligaments. The compositionsherein may provide an environment to attract tendon- or ligament-formingcells, stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions herein may also beuseful in the treatment of tendinitis, carpal tunnel syndrome, and othertendon or ligament defects. The compositions may also include anappropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

[0752] The PRO polypeptide or its agonist or antagonist may also beuseful for proliferation of neural cells and for regeneration of nerveand brain tissue, i.e., for the treatment of central and peripheralnervous system disease and neuropathies, as well as mechanical andtraumatic disorders, that involve degeneration, death, or trauma toneural cells or nerve tissue. More specifically, a PRO polypeptide orits agonist or antagonist may be used in the treatment of diseases ofthe peripheral nervous system, such as peripheral nerve injuries,peripheral neuropathy and localized neuropathies, and central nervoussystem diseases, such as Alzheimer's, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Furtherconditions that may be treated in accordance with the present inventioninclude mechanical and traumatic disorders, such as spinal corddisorders, head trauma, and cerebrovascular diseases such as stroke.Peripheral neuropathies resulting from chemotherapy or other medicaltherapies may also be treatable using a PRO polypeptide herein oragonist or antagonist thereto.

[0753] Ischemia-reperfusion injury is another indication. Endothelialcell dysfunction may be important in both the initiation of, and inregulation of the sequelae of events that occur followingischemia-reperfusion injury.

[0754] Rheumatoid arthritis is a further indication. Blood vessel growthand targeting of inflammatory cells through the vasculature is animportant component in the pathogenesis of rheumatoid and sero-negativeforms of arthritis.

[0755] A PRO polypeptide or its agonist or antagonist may also beadministered prophylactically to patients with cardiac hypertrophy, toprevent the progression of the condition, and avoid sudden death,including death of asymptomatic patients. Such preventative therapy isparticularly warranted in the case of patients diagnosed with massiveleft ventricular cardiac hypertrophy (a maximal wall thickness of 35 mmor more in adults, or a comparable value in children), or in instanceswhen the hemodynamic burden on the heart is particularly strong.

[0756] A PRO polypeptide or its agonist or antagonist may also be usefulin the management of atrial fibrillation, which develops in asubstantial portion of patients diagnosed with hypertrophiccardiomyopathy.

[0757] Further indications include angina, myocardial infarctions suchas acute myocardial infarctions, and heart failure such as congestiveheart failure. Additional non-neoplastic conditions include psoriasis,diabetic and other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, thyroidhyperplasias (including Grave's disease), corneal and other tissuetransplantation, chronic inflammation, lung inflammation, nephroticsyndrome, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

[0758] In view of the above, the PRO polypeptides or agonists orantagonists thereof described herein, which are shown to alter or impactendothelial cell function, proliferation, and/or form, are likely toplay an important role in the etiology and pathogenesis of many or allof the disorders noted above, and as such can serve as therapeutictargets to augment or inhibit these processes or for vascular-relateddrug targeting in these disorders.

[0759] 5.2.4.11. Administration Protocols, Schedules, Doses, andFormulations

[0760] The molecules herein and agonists and antagonists thereto arepharmaceutically useful as a prophylactic and therapeutic agent forvarious disorders and diseases as set forth above.

[0761] Therapeutic compositions of the PRO polypeptides or agonists orantagonists are prepared for storage by mixing the desired moleculehaving the appropriate degree of purity with optional pharmaceuticallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

[0762] Additional examples of such carriers include ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, and polyethyleneglycol. Carriers for topical or gel-based forms of agonist or antagonistinclude polysaccharides such as sodium carboxymethylcellulose ormethylcellulose, polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols. For all administrations, conventional depot formsare suitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations. The PROpolypeptides or agonists or antagonists will typically be formulated insuch vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml.

[0763] Another formulation comprises incorporating a PRO polypeptide oragonist or antagonist thereof into formed articles. Such articles can beused in modulating endothelial cell growth and angiogenesis. Inaddition, tumor invasion and metastasis may be modulated with thesearticles.

[0764] PRO polypeptides or agonists or antagonists to be used for invivo administration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. PRO polypeptides ordinarily will bestored in lyophilized form or in solution if administered systemically.If in lyophilized form, the PRO polypeptide or agonist or antagonistthereto is typically formulated in combination with other ingredientsfor reconstitution with an appropriate diluent at the time for use. Anexample of a liquid formulation of a PRO polypeptide or agonist orantagonist is a sterile, clear, colorless unpreserved solution filled ina single-dose vial for subcutaneous injection. Preserved pharmaceuticalcompositions suitable for repeated use may contain, for example,depending mainly on the indication and type of polypeptide:

[0765] a) PRO polypeptide or agonist or antagonist thereto;

[0766] b) a buffer capable of maintaining the pH in a range of maximumstability of the polypeptide or other molecule in solution, preferablyabout 4-8;

[0767] c) a detergent/surfactant primarily to stabilize the polypeptideor molecule against agitation-induced aggregation;

[0768] d) an isotonifier;

[0769] e) a preservative selected from the group of phenol, benzylalcohol and a benzethonium halide, e.g., chloride; and

[0770] f) water.

[0771] If the detergent employed is non-ionic, it may, for example, bepolysorbates (e.g., POLYSORBATE™ (TWEEN™) 20, 80, etc.) or poloxamers(e.g., POLOXAMER™ 188). The use of non-ionic surfactants permits theformulation to be exposed to shear surface stresses without causingdenaturation of the polypeptide. Further, such surfactant-containingformulations may be employed in aerosol devices such as those used in apulmonary dosing, and needleless jet injector guns (see, e.g., EP257,956).

[0772] An isotonifier may be present to ensure isotonicity of a liquidcomposition of the PRO polypeptide or agonist or antagonist thereto, andincludes polyhydric sugar alcohols, preferably trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, andmannitol. These sugar alcohols can be used alone or in combination.Alternatively, sodium chloride or other appropriate inorganic salts maybe used to render the solutions isotonic.

[0773] The buffer may, for example, be an acetate, citrate, succinate,or phosphate buffer depending on the pH desired. The pH of one type ofliquid formulation of this invention is buffered in the range of about 4to 8, preferably about physiological pH.

[0774] The preservatives phenol, benzyl alcohol and benzethoniumhalides, e.g., chloride, are known antimicrobial agents that may beemployed.

[0775] Therapeutic PRO polypeptide compositions generally are placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle. The formulations are preferablyadministered as repeated intravenous (i.v.), subcutaneous (s.c.), orintramuscular (i.m.) injections, or as aerosol formulations suitable forintranasal or intrapulmonary delivery (for intrapulmonary delivery see,e.g., EP 257,956).

[0776] PRO polypeptides can also be administered in the form ofsustained-released preparations. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the protein, which matrices are in the form ofshaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (e.g.,poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J.Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)), polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556(1983)),non-degradable ethylene-vinyl acetate (Langer et al., supra), degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid (EP133,988).

[0777] While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated proteins remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for proteinstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0778] Sustained-release PRO polypeptide compositions also includeliposomally entrapped PRO polypeptides. Liposomes containing the PROpolypeptide are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al,Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese patent application83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. % cholesterol, the selected proportion being adjusted for theoptimal therapy.

[0779] The therapeutically effective dose of a PRO polypeptide oragonist or antagonist thereto will, of course, vary depending on suchfactors as the pathological condition to be treated (includingprevention), the method of administration, the type of compound beingused for treatment, any co-therapy involved, the patient's age, weight,general medical condition, medical history, etc., and its determinationis well within the skill of a practicing physician. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the maximal therapeutic effect.If the PRO polypeptide has a narrow host range, for the treatment ofhuman patients formulations comprising human PRO polypeptide, morepreferably native-sequence human PRO polypeptide, are preferred. Theclinician will administer the PRO polypeptide until a dosage is reachedthat achieves the desired effect for treatment of the condition inquestion. For example, if the objective is the treatment of CHF, theamount would be one that inhibits the progressive cardiac hypertrophyassociated with this condition. The progress of this therapy is easilymonitored by echo cardiography. Similarly, in patients with hypertrophiccardiomyopathy, the PRO polypeptide can be administered on an empiricalbasis.

[0780] With the above guidelines, the effective dose generally is withinthe range of from about 0.001 to about 1.0 mg/kg, more preferably about0.01-1.0 mg/kg, most preferably about 0.01-0.1 mg/kg.

[0781] For non-oral use in treating human adult hypertension, it isadvantageous to administer the PRO polypeptide in the form of aninjection at about 0.01 to 50 mg, preferably about 0.05 to 20 mg, mostpreferably 1 to 20 mg, per kg body weight, 1 to 3 times daily byintravenous injection. For oral administration, a molecule based on thePRO polypeptide is preferably administered at about 5 mg to 1 g,preferably about 10 to 100 mg, per kg body weight, 1 to 3 times daily.It should be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less than 0.5 ng/mg protein.Moreover, for human administration, the formulations preferably meetsterility, pyrogenicity, general safety, and purity as required by FDAOffice and Biologics standards.

[0782] The dosage regimen of a pharmaceutical composition containing thePRO polypeptide to be used in tissue regeneration will be determined bythe attending physician considering various factors that modify theaction of the polypeptides, e.g., amount of tissue weight desired to beformed, the site of damage, the condition of the damaged tissue, thesize of a wound, type of damaged tissue (e.g., bone), the patient's age,sex, and diet, the severity of any infection, time of administration,and other clinical factors. The dosage may vary with the type of matrixused in the reconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF-I, to the final composition may also affectthe dosage. Progress can be monitored by periodic assessment oftissue/bone growth and/or repair, for example, X-rays, histomorphometricdeterminations, and tetracycline labeling.

[0783] The route of PRO polypeptide or antagonist or agonistadministration is in accord with known methods, e.g., by injection orinfusion by intravenous, intramuscular, intracerebral, intraperitoneal,intracerobrospinal, subcutaneous, intraocular, intraarticular,intrasynovial, intrathecal, oral, topical, or inhalation routes, or bysustained-release systems as noted below. The PRO polypeptide or agonistor antagonists thereof also are suitably administered by intratumoral,peritumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. The intraperitoneal route isexpected to be particularly useful, for example, in the treatment ofovarian tumors.

[0784] If a peptide or small molecule is employed as an antagonist oragonist, it is preferably administered orally or non-orally in the formof a liquid or solid to mammals.

[0785] Examples of pharmacologically acceptable salts of molecules thatform salts and are useful hereunder include alkali metal salts (e.g.,sodium salt, potassium salt), alkaline earth metal salts (e.g., calciumsalt, magnesium salt), ammonium salts, organic base salts (e.g.,pyridine salt, triethylamine salt), inorganic acid salts (e.g.,hydrochloride, sulfate, nitrate), and salts of organic acid (e.g.,acetate, oxalate, p-toluenesulfonate).

[0786] For compositions herein that are useful for bone, cartilage,tendon, or ligament regeneration, the therapeutic method includesadministering the composition topically, systemically, or locally as animplant or device. When administered, the therapeutic composition foruse is in a pyrogen-free, physiologically acceptable form. Further, thecomposition may desirably be encapsulated or injected in a viscous formfor delivery to the site of bone, cartilage, or tissue damage. Topicaladministration may be suitable for wound healing and tissue repair.

[0787] Preferably, for bone and/or cartilage formation, the compositionwould include a matrix capable of delivering the protein-containingcomposition to the site of bone and/or cartilage damage, providing astructure for the developing bone and cartilage and preferably capableof being resorbed into the body. Such matrices may be formed ofmaterials presently in use for other implanted medical applications.

[0788] The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance, andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid,polyglycolic acid, and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above-mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability.

[0789] One specific embodiment is a 50:50 (mole weight) copolymer oflactic acid and glycolic acid in the form of porous particles havingdiameters ranging from 150 to 800 microns. In some applications, it willbe useful to utilize a sequestering agent, such as carboxymethylcellulose or autologous blood clot, to prevent the polypeptidecompositions from disassociating from the matrix.

[0790] One suitable family of sequestering agents is cellulosicmaterials such as alkylcelluloses (including hydroxyalkylcelluloses),including methylcellulose, ethylcellulose, hydoxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, andcarboxymethylcellulose, one preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer, and poly(vinyl alcohol).The amount of sequestering agent useful herein is 0.5-20 wt %,preferably 1-10 wt %, based on total formulation weight, whichrepresents the amount necessary to prevent desorption of the polypeptide(or its antagonist) from the polymer matrix and to provide appropriatehandling of the composition, yet not so much that the progenitor cellsare prevented from infiltrating the matrix, thereby providing thepolypeptide (or its antagonist) the opportunity to assist the osteogenicactivity of the progenitor cells.

[0791] 5.2.4.12. Combination Therapies

[0792] The effectiveness of the PRO polypeptide or an agonist orantagonist thereof in preventing or treating the disorder in questionmay be improved by administering the active agent serially or incombination with another agent that is effective for those purposes,either in the same composition or as separate compositions.

[0793] For example, for treatment of cardiac hypertrophy, PROpolypeptide therapy can be combined with the administration ofinhibitors of known cardiac myocyte hypertrophy factors, e.g.,inhibitors of α-adrenergic agonists such as phenylephrine; endothelin-1inhibitors such as BOSENTAN™ and MOXONODIN™; inhibitors to CT-1 (U.S.Pat. No. 5,679,545); inhibitors to LIF; ACE inhibitors;des-aspartate-angiotensin I inhibitors (U.S. Pat. No. 5,773,415), andangiotensin II inhibitors.

[0794] For treatment of cardiac hypertrophy associated withhypertension, the PRO polypeptide can be administered in combinationwith α-adrenergic receptor blocking agents, e.g., propranolol, timolol,tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol,atenolol, metoprolol, or carvedilol; ACE inhibitors, e.g., quinapril,captopril, enalapril, ramipril, benazepril, fosinopril, or lisinopril;diuretics, e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, orindapamide; and/or calcium channel blockers, e.g., diltiazem,nifedipine, verapamil, or nicardipine. Pharmaceutical compositionscomprising the therapeutic agents identified herein by their genericnames are commercially available, and are to be administered followingthe manufacturers' instructions for dosage, administration, adverseeffects, contraindications, etc. See, e.g., Physicians' Desk Reference(Medical Economics Data Production Co.: Montvale, N.J., 1997), 51thEdition.

[0795] Preferred candidates for combination therapy in the treatment ofhypertrophic cardiomyopathy are β-adrenergic-blocking drugs (e.g.,propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol,penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol),verapamil, difedipine, or diltiazem. Treatment of hypertrophy associatedwith high blood pressure may require the use of antihypertensive drugtherapy, using calcium channel blockers, e.g., diltiazem, nifedipine,verapamil, or nicardipine; α-adrenergic blocking agents; diuretics,e.g., chlorothiazide, hydrochlorothiazide, hydroflumethazide,methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, orindapamide; and/or ACE-inhibitors, e.g., quinapril, captopril,enalapril, ramipril, benazepril, fosinopril, or lisinopril.

[0796] For other indications, PRO polypeptides or their agonists orantagonists may be combined with other agents beneficial to thetreatment of the bone and/or cartilage defect, wound, or tissue inquestion. These agents include various growth factors such as EGF, PDGF,TGF-α or TGF-β, IGF, FGF, and CTGF.

[0797] In addition, PRO polypeptides or their agonists or antagonistsused to treat cancer may be combined with cytotoxic, chemotherapeutic,or growth-inhibitory agents as identified above. Also, for cancertreatment, the PRO polypeptide or agonist or antagonist thereof issuitably administered serially or in combination with radiologicaltreatments, whether involving irradiation or administration ofradioactive substances.

[0798] The effective amounts of the therapeutic agents administered incombination with the PRO polypeptide or agonist or antagonist thereofwill be at the physician's or veterinarian's discretion. Dosageadministration and adjustment is done to achieve maximal management ofthe conditions to be treated. For example, for treating hypertension,these amounts ideally take into account use of diuretics or digitalis,and conditions'such as hyper- or hypotension, renal impairment, etc. Thedose will additionally depend on such factors as the type of thetherapeutic agent to be used and the specific patient being treated.Typically, the amount employed will be the same dose as that used, ifthe given therapeutic agent is administered without the PRO polypeptide.

[0799]5.2.4.13. Articles of Manufacture

[0800] An article of manufacture such as a kit containing the PROpolypeptide or agonists or antagonists thereof useful for the diagnosisor treatment of the disorders described above comprises at least acontainer and a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition that is effective for diagnosing or treating thecondition and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The active agent in the compositionis the PRO polypeptide or an agonist or antagonist thereto. The labelon, or associated with, the container indicates that the composition isused for diagnosing or treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use. The article of manufacture may also comprisea second or third container with another active agent as describedabove.

[0801] 5.2.5. Antibodies

[0802] Some of the most promising drug candidates according to thepresent invention are antibodies and antibody fragments that may inhibitthe production or the gene product of the genes identified herein and/orreduce the activity of the gene products.

[0803] 5.2.5.1. Polyclonal Antibodies

[0804] Methods of preparing polyclonal antibodies are known to theskilled artisan. Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, ifdesired, an adjuvant. Typically, the immunizing agent and/or adjuvantwill be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include the PROpolypeptide or a fusion protein thereof. It may be useful to conjugatethe immunizing agent to a protein known to be immunogenic in the mammalbeing immunized. Examples of such immunogenic proteins include, but arenot limited to, keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants thatmay be employed include Freund's complete adjuvant and MPL-TDM adjuvant(monophosphoryl Lipid A or synthetic trehalose dicorynomycolate). Theimmunization protocol may be selected by one skilled in the art withoutundue experimentation.

[0805] 5.2.5.2. Monoclonal Antibodies

[0806] The anti-PRO antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

[0807] The immunizing agent will typically include the PRO polypeptideor a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice(New York: Academic Press, 1986), pp. 59-103. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine, and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

[0808] Preferred immortalized cell lines are those that fuseefficiently, support stable high-level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications (MarcelDekker, Inc.: New York, 1987) pp. 51-63.

[0809] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst the PRO polypeptide. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0810] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods. Goding, supra. Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

[0811] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0812] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy- andlight-chain constant domains in place of the homologous murine sequences(U.S. Pat. No. 4,816,567; Morrison et al., supra) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0813] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fe region so as to prevent heavy-chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0814] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly Fab fragments, can be accomplished using routine techniquesknown in the art.

[0815] 5.2.5.3. Human and Humanized Antibodies

[0816] The anti-PRO antibodies may further comprise humanized antibodiesor human antibodies. Humanized forms of non-human (e.g., murine)antibodies are chimeric immunoglobulins, immunoglobulin chains, orfragments thereof (such as Fv, Fab, Fab′, F(ab′)₂, or otherantigen-binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a CDR of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin, and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody preferably also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. Jones et al., Nature, 321: 522-525(1986); Riechmann et al., Nature, 332: 323-329 (1988); Presta, Curr. Op.Struct. Biol., 2:593-596 (1992).

[0817] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al, Nature, 321: 522-525 (1986); Riechmann et al., Nature,332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)),by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0818] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries. Hoogenboom andWinter, J. Mol. Biol., 227: 381(1991); Marks et al., J. Mol. Biol., 222:581(1991). The techniques of Cole et al. and Boemer et al. are alsoavailable for the preparation of human monoclonal antibodies. Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1): 86-95 (1991). Similarly,human antibodies can be made by introducing human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed that closely resemblesthat seen in humans in all respects, including gene rearrangement,assembly, and antibody repertoire. This approach is described, forexample, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016, and in the following scientific publications:Marks et al., Bio/Technology, 10: 779-783 (1992); Lonberg et al.,Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994);Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger,Nature Biotechnology, 14: 826 (1996); Lonberg and Huszar, Intern. Rev.Immunol., 13: 65-93 (1995).

[0819] 5.2.5.4. Bispecific Antibodies

[0820] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the PRO polypeptide, the other one is for any otherantigen, and preferably for a cell-surface protein or receptor orreceptor subunit.

[0821] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities. Milsteinand Cuello, Nature, 305: 537-539 (1983). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655-3659(1991).

[0822] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant-domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH 1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies, see, for example,Suresh et al., Methods in Enzymology, 121: 210 (1986).

[0823] 5.2.5.5. Heteroconjugate Antibodies

[0824] Heteroconjugate antibodies are composed of two covalently joinedantibodies. Such antibodies have, for example, been proposed to targetimmune-system cells to unwanted cells (U.S. Pat. No. 4,676,980), and fortreatment of HIV infection. WO 91/00360; WO 92/200373; EP 03089. It iscontemplated that the antibodies may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

[0825] 5.2.5.6. Effector Function Engineering

[0826] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance, e.g., the effectivenessof the antibody in treating cancer. For example, cysteine residue(s) maybe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See, Caron et al., J. Exp. Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See, Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

[0827] 5.2.5.7. Immunoconjugates

[0828] The invention also pertains to immunoconjugates comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate).

[0829] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fiordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

[0830] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, WO94/11026.

[0831] In another embodiment, the antibody may be conjugated to a“receptor” (such as streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0832] 5.2.5.8. Immunoliposomes

[0833] The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0834] Particularly useful liposomes can be generated by thereverse-phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See, Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

[0835] 5.2.5.9. Pharmaceutical Compositions of Antibodies

[0836] Antibodies specifically binding a PRO polypeptide identifiedherein, as well as other molecules identified by the screening assaysdisclosed hereinbefore, can be administered for the treatment of variousdisorders as noted above and below in the form of pharmaceuticalcompositions.

[0837] If the PRO polypeptide is intracellular and whole antibodies areused as inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993).

[0838] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Alternatively, or in addition, the composition maycomprise an agent that enhances its function, such as, for example, acytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitoryagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

[0839] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

[0840] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0841] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0842] 5.2.5.10. Methods of Treatment Using the Antibody

[0843] It is contemplated that the antibodies to a PRO polypeptide maybe used to treat various cardiovascular, endothelial, and angiogenicconditions as noted above.

[0844] The antibodies are administered to a mammal, preferably a human,in accord with known methods, such as intravenous administration as abolus or by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous administration of the antibody is preferred.

[0845] Other therapeutic regimens may be combined with theadministration of the antibodies of the instant invention as notedabove. For example, if the antibodies are to treat cancer, the patientto be treated with such antibodies may also receive radiation therapy.Alternatively, or in addition, a chemotherapeutic agent may beadministered to the patient. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service, Ed., M. C. Perry (Williams & Wilkins:Baltimore, Md., 1992). The chemotherapeutic agent may precede, or followadministration of the antibody, or may be given simultaneouslytherewith. The antibody may be combined with an anti-estrogen compoundsuch as tamoxifen or EVISTA™ or an anti-progesterone such as onapristone(see, EP 616812) in dosages known for such molecules.

[0846] If the antibodies are used for treating cancer, it may bedesirable also to administer antibodies against other tumor-associatedantigens, such as antibodies that bind to one or more of the ErbB2,EGFR, ErbB3, ErbB4, or VEGF receptor(s). These also include the agentsset forth above. Also, the antibody is suitably administered serially orin combination with radiological treatments, whether involvingirradiation or administration of radioactive substances. Alternatively,or in addition, two or more antibodies binding the same or two or moredifferent antigens disclosed herein may be co-administered to thepatient. Sometimes, it may be beneficial also to administer one or morecytokines to the patient. In a preferred embodiment, the antibodiesherein are co-administered with a growth-inhibitory agent. For example,the growth-inhibitory agent may be administered first, followed by anantibody of the present invention. However, simultaneous administrationor administration of the antibody of the present invention first is alsocontemplated. Suitable dosages for the growth-inhibitory agent are thosepresently used and may be lowered due to the combined action (synergy)of the growth-inhibitory agent and the antibody herein.

[0847] In one embodiment, vascularization of tumors is attacked incombination therapy. The anti-PRO polypeptide antibody and anotherantibody (e.g., anti-VEGF) are administered to tumor-bearing patients attherapeutically effective doses as determined, for example, by observingnecrosis of the tumor or its metastatic foci, if any. This therapy iscontinued until such time as no further beneficial effect is observed orclinical examination shows no trace of the tumor or any metastatic foci.Then TNF is administered, alone or in combination with an auxiliaryagent such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody,heregulin, anti-heregulin antibody, D-factor, interleukin-1 (IL-1),interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor(GM-C SF), or agents that promote microvascular coagulation in tumors,such as anti-protein C antibody, anti-protein S antibody, or C4b bindingprotein (see, WO 91/01753, published Feb. 21, 1991), or heat orradiation.

[0848] Since the auxiliary agents will vary in their effectiveness, itis desirable to compare their impact on the tumor by matrix screening inconventional fashion. The administration of anti-PRO polypeptideantibody and TNF is repeated until the desired clinical effect isachieved. Alternatively, the anti-PRO polypeptide antibody isadministered together with TNF and, optionally auxiliary agent(s). Ininstances where solid tumors are found in the limbs or in otherlocations susceptible to isolation from the general circulation, thetherapeutic agents described herein are administered to the isolatedtumor or organ. In other embodiments, a FGF or PDGF antagonist, such asan anti-FGF or an anti-PDGF neutralizing antibody, is administered tothe patient in conjunction with the anti-PRO polypeptide antibody.Treatment with anti-PRO polypeptide antibodies preferably may besuspended during periods of wound healing or desirableneovascularization.

[0849] For the prevention or treatment of cardiovascular, endothelial,and angiogenic disorder, the appropriate dosage of an antibody hereinwill depend on the type of disorder to be treated, as defined above, theseverity and course of the disease, whether the antibody is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments.

[0850] For example, depending on the type and severity of the disorder,about 1 μg/kg to 50 mg/kg (e.g., 0. 1-20 mg/kg) of antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily or weekly dosage might range from about 1μg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is repeated or sustained until a desiredsuppression of disorder symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays, including, for example, radiographictumor imaging.

[0851]5.2.5.11. Articles of Manufacture with Antibodies

[0852] An article of manufacture containing a container with theantibody and a label is also provided. Such articles are describedabove, wherein the active agent is an anti-PRO antibody.

[0853] 5.2.5.12. Diagnosis and Prognosis of Tumors using Antibodies

[0854] If the indication for which the antibodies are used is cancer,while cell-surface proteins, such as growth receptors over expressed incertain tumors, are excellent targets for drug candidates or tumor(e.g., cancer) treatment, the same proteins along with PRO polypeptidesfind additional use in the diagnosis and prognosis of tumors. Forexample, antibodies directed against the PRO polypeptides may be used astumor diagnostics or prognostics.

[0855] For example, antibodies, including antibody fragments, can beused qualitatively or quantitatively to detect the expression of genesincluding the gene encoding the PRO polypeptide. The antibody preferablyis equipped with a detectable, e.g., fluorescent label, and binding canbe monitored by light microscopy, flow cytometry, fluorimetry, or othertechniques known in the art. Such binding assays are performedessentially as described above.

[0856] In situ detection of antibody binding to the marker gene productscan be performed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent to those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

[0857] The following Examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0858] The disclosures of all patent and literature references cited inthe present specification are hereby incorporated by reference in theirentirety.

6. EXAMPLES

[0859] Commercially available reagents referred to in the Examples wereused according to manufacturers instructions unless otherwise indicated.The source of those cells identified in the following Examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., supra; Ausubel et al., Current Protocols inMolecular Biology (Green Publishing Associates and Wiley Interscience,N.Y., 1989); Innis et al., PCR Protocols: A Guide to Methods andAplications (Academic Press, Inc.: N.Y., 1990); Harlow et al.,Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold SpringHarbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford,1984); Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

6.1. Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

[0860] The extracellular domain (ECD) sequences (including the secretionsignal sequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ®, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)) asa comparison of the ECD protein sequences to a 6 frame translation ofthe EST sequences. Those comparisons with a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

[0861] Using this extracellular domain homology screen, consensus DNAsequences were assembled relative to the other identified EST sequencesusing phrap. In addition, the consensus DNA sequences obtained wereoften (but not always) extended using repeated cycles of BLAST orBLAST-2 and phrap to extend the consensus sequence as far as possibleusing the sources of EST sequences discussed above.

[0862] Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

[0863] The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK⁵D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

6.2. Example 2 Isolation of cDNA Clones by Amylase Screening

[0864] 6.2.1. Preparation of Oligo dT Primed cDNA Library

[0865] mRNA was isolated from a human tissue of interest using reagentsand protocols from Invitrogen, San Diego, Calif. (Fast Track 2). ThisRNA was used to generate an oligo dT primed cDNA library in the vectorpRK5D using reagents and protocols from Life Technologies, Gaithersburg,Md. (Super Script Plasmid System). In this procedure, the doublestranded cDNA was sized to greater than 1000 bp and the SalI/NotIlinkered cDNA was cloned into XhoI/NotI cleaved vector. pRK5D is acloning vector that has an sp6 transcription initiation site followed byan SfiI restriction enzyme site preceding the Xhol/NotI cDNA cloningsites.

[0866] 6.2.2. Preparation of Random Primed cDNA Library

[0867] A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500-1000 bp, Tinkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

[0868] 6.2.3. Transformation and Detection

[0869] DNA from the library described in paragraph 2 above was chilledon ice to which was added electrocompetent DH10B bacteria (LifeTechnologies, 20 ml). The bacteria and vector mixture was thenelectroporated as recommended by the manufacturer. Subsequently, SOCmedia (Life Technologies, 1 ml) was added and the mixture was incubatedat 37° C. for 30 minutes. The transformants were then plated onto 20standard 150 mm LB plates containing ampicillin and incubated for 16hours (37° C.). Positive colonies were scraped off the plates and theDNA was isolated from the bacterial pellet using standard protocols,e.g., CsCl-gradient. The purified DNA was then carried on to the yeastprotocols below.

[0870] The yeast methods were divided into three categories: (1)Transformation of yeast with the plasmid/cDNA combined vector; (2)Detection and isolation of yeast clones secreting amylase; and (3) PCRamplification of the insert directly from the yeast colony andpurification of the DNA for sequencing and further analysis.

[0871] The yeast strain used was HD56-5A (ATCC-90785). This strain hasthe following genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

[0872] Transformation was performed based on the protocol outlined byGietz et al., Nucl. Acid. Res., 20:1425 (1992). Transformed cells werethen inoculated from agar into YEPD complex media broth (100 ml) andgrown overnight at 30° C. The YEPD broth was prepared as described inKaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., p. 207 (1994). The overnight culture was thendiluted to about 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPDbroth (500 ml) and regrown to ×10⁷ cells/ml (approx. OD₆₀₀=0.4-0.5).

[0873] The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

[0874] Transformation took place by mixing the prepared cells (100 μl)with freshly denatured single stranded salmon testes DNA (LofstrandLabs, Gaithersburg, Md.) and transforming DNA (1 μg, vol. <10 μl) inmicrofuge tubes. The mixture was mixed briefly by vortexing, then 40%PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA,100 mM Li₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes the reaction vessel centrifuged ina microfuge at 12,000 rpm for 5-10 seconds, decanted and resuspendedinto TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followed byrecentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

[0875] Alternatively, instead of multiple small reactions, thetransformation was performed using a single, large scale reaction,wherein reagent amounts were scaled up accordingly.

[0876] The selective media used was a synthetic complete dextrose agarlacking uracil (SCD-Ura) prepared as described in Kaiser et al., Methodsin Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,p. 208-210 (1994). Transformants were grown at 30° C. for 2-3 days.

[0877] The detection of colonies secreting amylase was performed byincluding red starch in the selective growth media. Starch was coupledto the red dye (Reactive Red-120, Sigma) as per the procedure describedby Biely et al., Anal. Biochem., 172:176-179 (1988). The coupled starchwas incorporated into the SCD-Ura agar plates at a final concentrationof 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50-100 mM final concentration).

[0878] The positive colonies were picked and streaked across freshselective media (onto 150 mm plates) in order to obtain well isolatedand identifiable single colonies. Well isolated single colonies positivefor amylase secretion were detected by direct incorporation of redstarch into buffered SCD-Ura agar. Positive colonies were determined bytheir ability to break down starch resulting in a clear halo around thepositive colony visualized directly.

[0879] 6.2.4. Isolation of DNA by PCR Amplification

[0880] When a positive colony was isolated, a portion of it was pickedby a toothpick and diluted into sterile water (30 μl) in a 96 wellplate. At this time, the positive colonies were either frozen and storedfor subsequent analysis or immediately amplified. An aliquot of cells (5μl) was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNTP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. Thesequence of the forward oligonucleotide 1 was:

[0881] 5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′ (SEQ ID NO:382)

[0882] The sequence of reverse oligonucleotide 2 was:

[0883] 5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′ (SEQ ID NO: 383)

[0884] PCR was then performed as follows: a. Denature 92° C.,  5 minutesb.  3 cycles of: Denature 92° C., 30 seconds Anneal 59° C., 30 secondsExtend 72° C., 60 seconds c.  3 cycles of: Denature 92° C., 30 secondsAnneal 57° C., 30 seconds Extend 72° C., 60 seconds d. 25 cycles of:Denature 92° C., 30 seconds Anneal 55° C., 30 seconds Extend 72° C., 60seconds e. Hold  4° C.

[0885] The underlined regions of the oligonucleotides annealed to theADH promoter region and the amylase region, respectively, and amplifieda 307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the, sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

[0886] Following the PCR, an aliquot of the reaction (5 μl) was examinedby agarose gel electrophoresis in a 1% agarose gel using aTris-Borate-EDTA (TBE) buffering system as described by Sambrook et al.,supra. Clones resulting in a single strong PCR product larger than 400bp were further analyzed by DNA sequencing after purification with a 96Qiaquick PCR clean-up column (Qiagen Inc., Chatsworth, Calif.).

6.3. Example 3 Isolation of cDNA Clones Using Signal Algorithm Analysis

[0887] Various polypeptide-encoding nucleic acid sequences wereidentified by applying a proprietary signal sequence finding algorithmdeveloped by Genentech, Inc., (South San Francisco, Calif.) upon ESTs aswell as clustered and assembled EST fragments from public (e.g.,GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals, Inc., PaloAlto, Calif.) databases. The signal sequence algorithm computes asecretion signal score based on the character of the DNA nucleotidessurrounding the first and optionally the second methionine codon(s)(ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals. Use of thisalgorithm resulted in the identification of numerouspolypeptide-encoding nucleic acid sequences.

6.4. Example 4 Isolation of cDNA Clones Encoding Human PRO Polypeptides

[0888] Using the techniques described in Examples 1 to 3 above, numerousfull-length cDNA clones were identified as encoding PRO polypeptides asdisclosed herein. These cDNAs were then deposited under the terms of theBudapest Treaty with the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, USA (ATCC) as shown in Table7 below. TABLE 7 Material ATCC Dep. No. Deposit Date 23330-1390 209775Apr. 14, 1998 23339-1130 209282 Sep. 18, 1997 26846-1397 203406 Oct. 27,1998 26847-1395 209772 Apr. 14, 1998 27865-1091 209296 Sep. 23, 199730868-1156 1437-PTA Mar. 2, 2000 30871-1157 209380 Oct. 16, 199732286-1191 209385 Oct. 16, 1997 33089-1132 209262 Sep. 16, 199733092-1202 209420 Oct. 28, 1997 33100-1159 209377 Oct. 16, 199733223-1136 209264 Sep. 16, 1997 34392-1170 209526 Dec. 10, 199734431-1177 209399 Oct. 17, 1997 34433-1308 209719 Mar. 31, 199834434-1139 209252 Sep. 16, 1997 35600-1162 209370 Oct. 16, 199735673-1201 209418 Oct. 28, 1997 35880-1160 209379 Oct. 16, 199735918-1174 209402 Oct. 17, 1997 36350-1158 209378 Oct. 16, 199736638-1056 209456 Nov. 12, 1997 38268-1188 209421 Oct. 28, 199740370-1217 209485 Nov. 21, 1997 40628-1216 209432 Nov. 7, 199743316-1237 209487 Nov. 21, 1997 44196-1353 209847 May 6, 1998 45409-2511203579 Jan. 12, 1999 45419-1252 209616 Feb. 5, 1998 46777-1253 209619Feb. 5, 1998 48336-1309 209669 Mar. 11, 1998 48606-1479 203040 Jul. 1,1998 49435-1219 209480 Nov. 21, 1997 49631-1328 209806 Apr. 28, 199850919-1361 209848 May 6, 1998 50920-1325 209700 Mar. 26, 1998 50921-1458209859 May 12, 1998 52758-1399 209773 Apr. 14, 1998 53517-1366-1 209802Apr. 23, 1998 53915-1258 209593 Jan. 21, 1998 53974-1401 209774 Apr. 14,1998 53987-1438 209858 May 12, 1998 56047-1456 209948 Jun. 9, 199856050-1455 203011 Jun. 23, 1998 56110-1437 203113 Aug. 11, 199856405-1357 209849 May 6, 1998 56433-1406 209857 May 12, 1998 56439-1376209864 May 12, 1998 56529-1647 203293 Sep. 29, 1998 56865-1491 203022Jun. 23, 1998 56965-1356 209842 May 6, 1998 57033-1403-1 209905 May 27,1998 57037-1444 209903 May 27, 1998 57039-1402 209777 Apr. 14, 199857689-1385 209869 May 14, 1998 57690-1374 209950 Jun. 9, 1998 57694-1341203017 Jun. 23, 1998 57695-1340 203006 Jun. 23, 1998 57699-1412 203020Jun. 23, 1998 57700-1408 203583 Jan. 12, 1999 57708-1411 203021 Jun. 23,1998 57838-1337 203014 Jun. 23, 1998 58847-1383 209879 May 20, 199858852-1637 203271 Sep. 22, 1998 58853-1423 203016 Jun. 23, 199859212-1627 203245 Sep. 9, 1998 59220-1514 209962 Jun. 9, 1998 59493-1420203050 Jul. 1, 1998 59497-1496 209941 Jun. 4, 1998 59586-1520 203288Sep. 29, 1998 59588-1571 203106 Aug. 11, 1998 59620-1463 209989 Jun. 16,1998 59622-1334 209984 Jun. 16, 1998 59777-1480 203111 Aug. 11, 199859848-1512 203088 Aug. 4, 1998 59849-1504 209986 Jun. 16, 199860621-1516 203091 Aug. 4, 1998 60622-1525 203090 Aug. 4, 1998 60764-1533203452 Nov. 10, 1998 60783-1611 203130 Aug. 18, 1998 61755-1554 203112Aug. 11, 1998 62306-1570 203254 Sep. 9, 1998 62312-2558 203836 Mar. 9,1999 62814-1521 203093 Aug. 4, 1998 62872-1509 203100 Aug. 4, 199864883-1526 203253 Sep. 9, 1998 64886-1601 203241 Sep. 9, 1998 64889-1541203250 Sep. 9, 1998 64896-1539 203238 Sep. 9, 1998 64897-1628 203216Sep. 15, 1998 64903-1553 203223 Sep. 15, 1998 64908-1163-1 203243 Sep.9, 1998 64950-1590 203224 Sep. 15, 1998 65402-1540 203252 Sep. 9, 199865404-1551 203244 Sep. 9, 1998 65405-1547 203476 Nov. 17, 199865410-1569 203231 Sep. 15, 1998 65412-1523 203094 Aug. 4, 199866307-2661  431-PTA Jul. 27, 1999 66526-1616 203246 Sep. 9, 199866659-1593 203269 Sep. 22, 1998 66660-1585 203279 Sep. 22, 199866667-1596 203267 Sep. 22, 1998 66672-1586 203265 Sep. 22, 199866675-1587 203282 Sep. 22, 1998 67300-1605 203163 Aug. 25, 199868818-2536 203657 Feb. 9, 1999 68862-2546 203652 Feb. 9, 1999 68872-1620203160 Aug. 25, 1998 71290-1630 203275 Sep. 22, 1998 73736-1657 203466Nov. 17, 1998 73739-1645 203270 Sep. 22, 1998 73742-1662 203316 Oct. 6,1998 76385-1692 203664 Feb. 9, 1999 76393-1664 203323 Oct. 6, 199876399-1700 203472 Nov. 17, 1998 76400-2528 203573 Jan. 12, 199976510-2504 203477 Nov. 17, 1998 76529-1666 203315 Oct. 6, 199876532-1702 203473 Nov. 17, 1998 76541-1675 203409 Oct. 27, 199877503-1686 203362 Oct. 20, 1998 77624-2515 203553 Dec, 22, 199879230-2525 203549 Dec. 22, 1998 79862-2522 203550 Dec. 22, 199880145-2594  204-PTA Jun. 8, 1999 80899-2501 203539 Dec. 15, 199881754-2532 203542 Dec. 15, 1998 81757-2512 203543 Dec. 15, 199881761-2583 203862 Mar. 23, 1999 82358-2738  510-PTA Aug. 10, 199982364-2538 203603 Jan. 20, 1999 82403-2959 2317-PTA Aug. 1, 200083500-2506 203391 Oct. 29, 1998 83560-2569 203816 Mar. 2, 199984210-2576 203818 Mar. 2, 1999 84920-2614 203966 Apr. 27, 199986576-2595 203868 Mar. 23, 1999 92218-2554 203834 Mar. 9, 199992233-2599  134-PTA May 25, 1999 92256-2596 203891 Mar. 30, 199992265-2669  256-PTA Jun. 22, 1999 92274-2617 203971 Apr. 27, 199992929-2534-1 203586 Jan. 12, 1999 93011-2637  20-PTA May 4, 199994854-2586 203864 Mar. 23, 1999 96787-2534-1 203589 Jan. 12, 199996867-2620 203972 Apr. 27, 1999 96872-2674  550-PTA Aug. 17, 199996878-2626  23-PTA May 4, 1999 96889-2641  119-PTA May 25, 1999100312-2645  44-PTA May 11, 1999 105782-2693  387-PTA Jul. 20, 1999105849-2704  473-PTA Aug. 3, 1999 108725-2766  863-PTA Oct. 19, 1999108769-2765  861-PTA Oct. 19, 1999 119498-2965 2298-PTA Jul. 25, 2000119535-2756  613-PTA Aug. 31, 1999 125185-2806 1031-PTA Dec. 7, 1999131639-2874 1784-PTA Apr. 25, 2000 139623-2893 1670-PTA Apr. 11, 2000143076-2787 1028-PTA Dec. 7, 1999 143276-2975 2387-PTA Aug. 8, 2000164625-2890 1535-PTA Mar. 21, 2000 167678-2963 2302-PTA Jul. 25, 2000170021-2923 1906-PTA May 23, 2000 170212-3000 2583-PTA Oct. 10, 2000177313-2982 2251-PTA Jul. 19, 2000

[0889] These deposits were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of a viable culture of thedeposit for 30 years from the date of deposit. The deposits will be madeavailable by ATCC under the terms of the Budapest Treaty, and subject toan agreement between Genentech, Inc. and ATCC, which assures permanentand unrestricted availability of the progeny of the culture of thedeposit to the public upon issuance of the pertinent U.S. patent or uponlaying open to the public of any U.S. or foreign patent application,whichever comes first, and assures availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 USC § 122 and the Commissioner's rulespursuant thereto (including 37 CFR § 1.14 with particular reference to886 OG 638).

[0890] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

6.5 Example 5 Isolation of cDNA Clones Encoding Human PRO1873, PRO7223,PRO7248, PRO730. PRO532, PRO7261, PRO734, PRO771, PRO2010, PRO5723,PRO3444. PRO9940, PRO3562, PRO10008, PRO5730, PRO6008, PRO4527. PRO4538and PRO4553

[0891] DNA molecules encoding the PRO1873, PRO7223, PRO7248, PRO730,PRO532, PRO7261, PRO734, PRO771, PRO2010, PRO5723, PRO3444, PRO9940,PRO3562, PRO10008, PRO5730, PRO6008, PRO4527, PRO4538 and PRO4553polypeptides shown in the accompanying figures were obtained throughGenBank.

6.6. Example 6 Use of PRO as a Hybridization Probe

[0892] The following method describes use of a nucleotide sequenceencoding PRO as a hybridization probe.

[0893] DNA comprising the coding sequence of full-length or mature PRO(as shown in accompanying figures) or a fragment thereof is employed asa probe to screen for homologous DNAs (such as those encodingnaturally-occurring variants of PRO) in human tissue cDNA libraries orhuman tissue genomic libraries.

[0894] Hybridization and washing of filters containing either libraryDNAs is performed under the following high-stringency conditions.Hybridization of radiolabeled probe derived from the gene encoding PROpolypeptide to the filters is performed in a solution of 50% formamide,5×SSC, 0. 1% SDS, 0. 1% sodium pyrophosphate, 50 mM sodium phosphate, pH6.8, 2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20hours. Washing of the filters is performed in an aqueous solution of0.1×SSC and 0.1% SDS at 42° C.

[0895] DNAs having a desired sequence identity with the DNA encodingfull-length native sequence can then be identified using standardtechniques known in the art.

6.7. Example 7 Expression of PRO in E. coli

[0896] This example illustrates preparation of an unglycosylated form ofPRO by recombinant expression in E. coli.

[0897] The DNA sequence encoding PRO is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; see,Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apoly-His leader (including the first six STII codons, poly-His sequence,and enterokinase cleavage site), the PRO coding region, lambdatranscriptional terminator, and an argU gene.

[0898] The ligation mixture is then used to transform a selected E. colistrain using the methods described in Sambrook et al., supra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

[0899] Selected clones can be grown overnight in liquid culture mediumsuch as LB broth supplemented with antibiotics. The overnight culturemay subsequently be used to inoculate a larger scale culture. The cellsare then grown to a desired optical density, during which the expressionpromoter is turned on.

[0900] After culturing the cells for several more hours, the cells canbe harvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

[0901] PRO may be expressed in E. coli in a poly-His tagged form, usingthe following procedure. The DNA encoding PRO is initially amplifiedusing selected PCR primers. The primers will contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an OD₆₀₀ of 3-5 is reached. Cultures are thendiluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate•2H₂O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 sheffield hycase SF in 500 ml water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

[0902]E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1 M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi²⁺-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

[0903] The proteins are refolded by diluting the sample slowly intofreshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA.Refolding volumes are chosen so that the final protein concentration isbetween 50 to 100 micrograms/ml. The refolding solution is stirredgently at 4° C. for 12-36 hours. The refolding reaction is quenched bythe addition of TFA to a final concentration of 0.4% (pH ofapproximately 3). Before further purification of the protein, thesolution is filtered through a 0.22 micron filter and acetonitrile isadded to 2-10% final concentration. The refolded protein ischromatographed on a Poros R1/H reversed phase column using a mobilebuffer of 0.1% TFA with elution with a gradient of acetonitrile from 10to 80%. Aliquots of fractions with A₂₈₀ absorbance are analyzed on SDSpolyacrylamide gels and fractions containing homogeneous refoldedprotein are pooled. Generally, the properly refolded species of mostproteins are eluted at the lowest concentrations of acetonitrile sincethose species are the most compact with their hydrophobic interiorsshielded from interaction with the reversed phase resin. Aggregatedspecies are usually eluted at higher acetonitrile concentrations. Inaddition to resolving misfolded forms of proteins from the desired form,the reversed phase step also removes endotoxin from the samples.

[0904] Fractions containing the desired folded PRO polypeptide arepooled and the acetonitrile removed using a gentle stream of nitrogendirected at the solution. Proteins are formulated into 20 mM Hepes, pH6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gelfiltration using G25 Superfine (Pharmacia) resins equilibrated in theformulation buffer and sterile filtered.

[0905] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

6.8. Example 8 Expression of PRO in Mammalian Cells

[0906] This example illustrates preparation of a potentiallyglycosylated form of PRO by recombinant expression in mammalian cells.

[0907] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), isemployed as the expression vector. Optionally, the PRO DNA is ligatedinto pRK5 with selected restriction enzymes to allow insertion of thePRO DNA using ligation methods such as described in Sambrook et al.,supra. The resulting vector is called pRK5-PRO.

[0908] In one embodiment, the selected host cells may be 293 cells.Human 293 cells (ATCC CCL 1573) are grown to confluence in tissueculture plates in medium such as DMEM supplemented with fetal calf serumand optionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

[0909] Approximately 24 hours after the transfections, the culturemedium is removed and replaced with culture medium (alone) or culturemedium containing 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of the PRO polypeptide. The culturescontaining transfected cells may undergo further incubation (in serumfree medium) and the medium is tested in selected bioassays.

[0910] In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing expressed PRO can then be concentrated andpurified by any selected method, such as dialysis and/or columnchromatography.

[0911] In another embodiment, PRO can be expressed in CHO cells. ThepRK5-PRO can be transfected into CHO cells using known reagents such asCaPO₄ or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of a PRO polypeptide, the culture medium may be replaced withserum free medium. Preferably, the cultures are incubated for about 6days, and then the conditioned medium is harvested. The mediumcontaining the expressed PRO polypeptide can then be concentrated andpurified by any selected method.

[0912] Epitope-tagged PRO may also be expressed in host CHO cells. ThePRO may be subcloned out of the pRK5 vector. The subclone insert canundergo PCR to fuse in frame with a selected epitope tag such as apoly-His tag into a Baculovirus expression vector. The poly-His taggedPRO insert can then be subcloned into a SV40 driven vector containing aselection marker such as DHFR for selection of stable clones. Finally,the CHO cells can be transfected (as described above) with the SV40driven vector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedPRO can then be concentrated and purified by any selected method, suchas by Ni²⁺-chelate affinity chromatography.

[0913] PRO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

[0914] Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g., extracellular domains) of the respective proteins are fused to anIgG 1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or as a poly-His tagged form.

[0915] Following PCR amplification, the respective DNAs are subcloned ina CHO expression vector using standard techniques as described inAusubel et al., Current Protocols of Molecular Biology, Unit 3.16, JohnWiley and Sons (1997). CHO expression vectors are constructed to havecompatible restriction sites 5′ and 3′ of the DNA of interest to allowthe convenient shuttling of cDNA's. The vector used in expression in CHOcells is as described in Lucas et al., Nucl. Acids Res., 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

[0916] Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Qiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁷ cells are frozen in an ampule for furthergrowth and production as described below.

[0917] The ampules containing the plasmid DNA are thawed by placementinto a water bath and mixed by vortexing. The contents are pipetted intoa centrifuge tube containing 10 ml of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 ml of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 ml spinner containing 90 ml of selective media. After 1-2days, the cells are transferred into a 250 ml spinner filled with 150 mlselective growth medium and incubated at 37° C. After another 2-3 days,250 ml, 500 ml and 2000 ml spinners are seeded with 3×10⁵ cells/ml. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3L production spinner isseeded at 1.2×10⁶ cells/ml. On day 0, the cell number and pH isdetermined. On day 1, the spinner is sampled and sparging with filteredair is commenced. On day 2, the spinner is sampled, the temperatureshifted to 33° C., and ml of 500 g/L glucose and 0.6 ml of 10% antifoam(e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability drops below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate iseither stored at 4° C. or immediately loaded onto columns forpurification.

[0918] For the poly-His tagged constructs, the proteins are purifiedusing a Ni²⁺-NTA column (Qiagen). Before purification, imidazole isadded to the conditioned media to a concentration of 5 mM. Theconditioned media is pumped onto a 6 ml Ni²⁺-NTA column equilibrated in20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole ata flow rate of 4-5 ml/min. at 4° C. After loading, the column is washedwith additional equilibration buffer and the protein eluted withequilibration buffer containing 0.25 M imidazole. The highly purifiedprotein is subsequently desalted into a storage buffer containing 10 mMHepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine(Pharmacia) column and stored at −80° C.

[0919] Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which has been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μl of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

[0920] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

6.9. Example 9 Expression of PRO in Yeast

[0921] The following method describes recombinant expression of PRO inyeast.

[0922] First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding PRO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO. For secretion, DNA encoding PRO can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO signal peptide or other mammalian signal peptide, or, for example, ayeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression of PRO.

[0923] Yeast cells, such as yeast strain AB110, can then be transformedwith the expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0924] Recombinant PRO can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing PRO may further be purified using selected columnchromatography resins.

[0925] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

6.10. Example 10 Expression of PRO in Baculovirus-infected Insect Cells

[0926] The following method describes recombinant expression inBaculovirus-infected insect cells.

[0927] The sequence coding for PRO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-His tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL 1393 (Novagen). Briefly, thesequence encoding PRO or the desired portion of the coding sequence ofPRO (such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular) is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

[0928] Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

[0929] Expressed poly-His tagged PRO can then be purified, for example,by Ni²⁺-chelate affinity chromatography as follows. Extracts areprepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 ml Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 ml, washedwith 25 ml of water and equilibrated with 25 ml of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 ml per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes nonspecifically bound protein. After reachingA₂₈₀baseline again, the column is developed with a 0 to 500 mM imidazolegradient in the secondary wash buffer. One ml fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO are pooled and dialyzed againstloading buffer.

[0930] Alternatively, purification of the IgG tagged (or Fc tagged) PROcan be performed using known chromatography techniques, including forinstance, Protein A or protein G column chromatography.

[0931] Following PCR amplification, the respective coding sequences aresubcloned into a baculovirus expression vector (pb.PH.IgG for IgGfusions and pb.PH.His.c for poly-His tagged proteins), and the vectorand Baculogold® baculovirus DNA (Pharmingen) are co-transfected into 105Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711), using Lipofectin(Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of thecommercially available baculovirus expression vector pVL1393(Pharmingen), with modified polylinker regions to include the His or Fctag sequences. The cells are grown in Hink's TNM-FH medium supplementedwith 10% FBS (Hyclone). Cells are incubated for 5 days at 28° C. Thesupernatant is harvested and subsequently used for the first viralamplification by infecting Sf9 cells in Hink's TNM-FH mediumsupplemented with 10% FBS at an approximate multiplicity of infection(MOI) of 10. Cells are incubated for 3 days at 28° C. The supernatant isharvested and the expression of the constructs in the baculovirusexpression vector is determined by batch binding of 1 ml of supernatantto 25 ml of Ni²⁺-NTA beads (QIAGEN) for histidine tagged proteins orProtein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteinsfollowed by SDS-PAGE analysis comparing to a known concentration ofprotein standard by Coomassie blue staining.

[0932] The first viral amplification supernatant is used to infect aspinner culture (500 ml) of Sf9 cells grown in ESF-921 medium(Expression Systems LLC) at an approximate MOI of 0.1. Cells areincubated for 3 days at 28° C. The supernatant is harvested andfiltered. Batch binding and SDS-PAGE analysis is repeated, as necessary,until expression of the spinner culture is confirmed.

[0933] The conditioned medium from the transfected cells (0.5 to 3 L) isharvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteinconstruct is purified using a Ni²⁺-NTA column (Qiagen). Beforepurification, imidazole is added to the conditioned media to aconcentration of 5 mM. The conditioned media is pumped onto a 6 mlNi²⁺-NTA column equilibrated in 20 mM Repes, pH 7.4, buffer containing0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 m/min. at 4° C.After loading, the column is washed with additional equilibration bufferand the protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein is subsequently desalted into astorage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at −80° C.

[0934] Immunoadhesin (Fc containing) constructs of proteins are purifiedfrom the conditioned media as follows.

[0935] The conditioned media is pumped onto a 5 ml Protein A column(Pharmacia) which has been equilibrated in 20 mM Na phosphate buffer, pH6.8. After loading, the column is washed extensively with equilibrationbuffer before elution with 100 mM citric acid, pH 3.5. The elutedprotein is immediately neutralized by collecting 1 ml fractions intotubes containing 275 ml of 1 M Tris buffer, pH 9. The highly purifiedprotein is subsequently desalted into storage buffer as described abovefor the poly-His tagged proteins. The homogeneity of the proteins isverified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminalamino acid sequencing by Edman degradation.

[0936] Alternatively, a modified baculovirus procedure may be usedincorporating high-5 cells. In this procedure, the DNA encoding thedesired sequence is amplified with suitable systems, such as Pfu(Stratagene), or fused upstream (5′-of) of an epitope tag contained witha baculovirus expression vector. Such epitope tags include poly-His tagsand immunoglobulin tags (like Fc regions of IgG). A variety of plasmidsmay be employed, including plasmids derived from commercially availableplasmids such as pIE1-1 (Novagen). The pIE1-1 and pIE1-2 vectors aredesigned for constitutive expression of recombinant proteins from thebaculovirus ie1 promoter in stably-transformed insect cells (1). Theplasmids differ only in the orientation of the multiple cloning sitesand contain all promoter sequences known to be important forie1-mediated gene expression in uninfected insect cells as well as thehr5 enhancer element. pIE1-1 and pIE1-2 include the translationinitiation site and can be used to produce fusion proteins. Briefly, thedesired sequence or the desired portion of the sequence (such as thesequence encoding the extracellular domain of a transmembrane protein)is amplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product is then digested with those selected restrictionenzymes and subcloned into the expression vector. For example,derivatives of pIE1-1 can include the Fc region of human IgG (pb.PH.IgG)or an 8 histidine (pb.PH.His) tag downstream (3′-of) the desiredsequence. Preferably, the vector construct is sequenced forconfirmation.

[0937] High-5 cells are grown to a confluency of 50% under theconditions of, 27° C., no CO₂, NO pen/strep. For each 150 mm plate, 30μg of pIE based vector containing the sequence is mixed with 1 mlEx-Cell medium (Media:Ex-Cell 401+1/100 L-Glu JRH Biosciences #14401-78P(note: this media is light sensitive)), and in a separate tube, 100 μlof CellFectin (CellFECTIN (GibcoBRL #10362-010) (vortexed to mix)) ismixed with 1 ml of Ex-Cell medium. The two solutions are combined andallowed to incubate at room temperature for 15 minutes. 8 ml of Ex-Cellmedia is added to the 2 ml of DNA/CellFECTIN mix and this is layered onhigh-5 cells that have been washed once with Ex-Cell media. The plate isthen incubated in darkness for 1 hour at room temperature. TheDNA/CellFECTIN mix is then aspirated, and the cells are washed once withEx-Cell to remove excess CellFECTIN, 30 ml of fresh Ex-Cell media isadded and the cells are incubated for 3 days at 28° C. The supernatantis harvested and the expression of the sequence in the baculovirusexpression vector is determined by batch binding of 1 ml of supernatentto 25 ml of Ni²⁺-NTA beads (QIAGEN) for histidine tagged proteins orProtein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteinsfollowed by SDS-PAGE analysis comparing to a known concentration ofprotein standard by Coomassie blue staining.

[0938] The conditioned media from the transfected cells (0.5 to 3 L) isharvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteincomprising the sequence is purified using a Ni²⁺-NTA column (Qiagen).Before purification, imidazole is added to the conditioned media to aconcentration of 5 mM. The conditioned media is pumped onto a 6 mlNi²⁺-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 48° C.After loading, the column is washed with additional equilibration bufferand the protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein is then subsequently desaltedinto a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4%mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

[0939] Immunoadhesin (Fe containing) constructs of proteins are purifiedfrom the conditioned media as follows. The conditioned media is pumpedonto a 5 ml Protein A column (Pharmacia) which had been equilibrated in20 mM Na phosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 ml of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity of the sequence is assessed by SDS polyacrylamide gels andby N-terminal amino acid sequencing by Edman degradation and otheranalytical procedures as desired or necessary.

[0940] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

6.11. Example 11 Preparation of Antibodies that Bind PRO

[0941] This example illustrates preparation of monoclonal antibodieswhich can specifically bind the PRO polypeptide or an epitope on the PROpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

[0942] Techniques for producing the monoclonal antibodies are known inthe art and are described, for instance, in Goding, supra. Immunogensthat may be employed include purified PRO, fusion proteins containingPRO, and cells expressing recombinant PRO on the cell surface. Selectionof the immunogen can be made by the skilled artisan without undueexperimentation.

[0943] Mice, such as Balbic, are immunized with the PRO immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

[0944] After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterm, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

[0945] The hybridoma cells will be screened in an ELISA for reactivityagainst PRO. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against PRO is within the skill in theart.

[0946] The positive hybridoma cells can be injected intraperitoneallyinto syngeneic Balbic mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

6.12. Example 12 Purification of PRO Polypeptides Using SpecificAntibodies

[0947] Native or recombinant PRO polypeptides may be purified by avariety of standard techniques in the art of protein purification. Forexample, pro-PRO polypeptide, mature PRO polypeptide, or pre-PROpolypeptide is purified by immunoaffinity chromatography usingantibodies specific for the PRO polypeptide of interest. In general, animmunoaffinity column is constructed by covalently coupling the anti-PROpolypeptide antibody to an activated chromatographic resin.

[0948] Polyclonal immunoglobulins are prepared from immune sera eitherby precipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSEM (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

[0949] Such an immunoaffinity column is utilized in the purification ofPRO polypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

[0950] A soluble PRO polypeptide-containing preparation is passed overthe immunoaffinity column, and the column is washed under conditionsthat allow the preferential absorbance of PRO polypeptide (e.g., highionic strength buffers in the presence of detergent). Then, the columnis eluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

6.13. Example 13 Drug Screening

[0951] This invention is particularly useful for screening compounds byusing PRO polypeptides or binding fragment thereof in any of a varietyof drug screening techniques. The PRO polypeptide or fragment employedin such a test may either be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. One methodof drug screening utilizes eukaryotic or prokaryotic host cells whichare stably transformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

[0952] Thus, the present invention provides methods of screening fordrugs or any other agents which can affect a PRO polypeptide-associateddisease or disorder. These methods comprise contacting such an agentwith an PRO polypeptide or fragment thereof and assaying (I) for thepresence of a complex between the agent and the PRO polypeptide orfragment, or (ii) for the presence of a complex between the PROpolypeptide or fragment and the cell, by methods well known in the art.In such competitive binding assays, the PRO polypeptide or fragment istypically labeled. After suitable incubation, free PRO polypeptide orfragment is separated from that present in bound form, and the amount offree or uncomplexed label is a measure of the ability of the particularagent to bind to PRO polypeptide or to interfere with the PROpolypeptide/cell complex.

[0953] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to apolypeptide and is described in detail in WO 84/03564, published on Sep.13, 1984. Briefly stated, large numbers of different small peptide testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. As applied to a PRO polypeptide, the peptide testcompounds are reacted with PRO polypeptide and washed. Bound PROpolypeptide is detected by methods well known in the art. Purified PROpolypeptide can also be coated directly onto plates for use in theaforementioned drug screening techniques. In addition, non-neutralizingantibodies can be used to capture the peptide and immobilize it on thesolid support.

[0954] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

6.14. Example 14 Rational Drug Design

[0955] The goal of rational drug design is to produce structural analogsof biologically active polypeptide of interest (i.e., a PRO polypeptide)or of small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (cf., Hodgson, Bio/Technology, 9: 19-21 (1991)).

[0956] In one approach, the three-dimensional structure of the PROpolypeptide, or of an PRO polypeptide-inhibitor complex, is determinedby x-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).

[0957] It is also possible to isolate a target-specific antibody,selected by functional assay, as described above, and then to solve itscrystal structure. This approach, in principle, yields a pharmacore uponwhich subsequent drug design can be based. It is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (anti-ids) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-ids would be expected to be an analog of the original receptor. Theanti-id could then be used to identify and isolate peptides from banksof chemically or biologically produced peptides. The isolated peptideswould then act as the pharmacore.

[0958] By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

6.15. Example 15 Stimulation of Endothelial Cell Proliferation (Assay 8)

[0959] This assay is designed to determine whether PRO polypeptides ofthe present invention show the ability to stimulate adrenal corticalcapillary endothelial cell (ACE) growth. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of conditions or disorders where angiogenesiswould be beneficial including, for example, wound healing, and the like(as would agonists of these PRO polypeptides). Antagonists of the PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of cancerous tumors.

[0960] Bovine adrenal cortical capillary endothelial (ACE) cells (fromprimary culture, maximum of 12-14 passages) were plated in 96-wellplates at 500 cells/well per 100 microliter. Assay media included lowglucose DMEM, 10% calf serum, 2 mM glutamine, and1×penicillin/streptomycin/fungizone. Control wells included thefollowing: (1) no ACE cells added; (2) ACE cells alone; (3) ACE cellsplus VEGF (5 ng/ml); and (4) ACE cells plus FGF (5 ng/ml). The controlor test sample, (in 100 microliter volumes), was then added to the wells(at dilutions of 1%, 0.1% and 0.01%, respectively). The cell cultureswere incubated for 6-7 days at 37° C./5% CO₂. After the incubation, themedia in the wells was aspirated, and the cells were washed 1× with PBS.An acid phosphatase reaction mixture (100 microliter; 0.1M sodiumacetate, pH 5.5, 0.1% Triton X-100, 10 mM p-nitrophenyl phosphate) wasthen added to each well. After a 2 hour incubation at 37° C., thereaction was stopped by addition of 10 microliters 1N NaOH. Opticaldensity (OD) was measured on a microplate reader at 405 nm.

[0961] The activity of a PRO polypeptide was calculated as the foldincrease in proliferation (as determined by the acid phosphataseactivity, OD 405 nm) relative to (1) cell only background, and (2)relative to maximum stimulation by VEGF. VEGF (at 3-10 ng/ml) and FGF(at 1-5 ng/ml) were employed as an activity reference for maximumstimulation. Results of the assay were considered “positive” if theobserved stimulation was ≧50% increase over background. VEGF (5 ng/ml)control at 1% dilution gave 1.24 fold stimulation; FGF (5 ng/ml) controlat 1% dilution gave 1.46 fold stimulation.

[0962] PRO21 tested positive in this assay.

6.16. Example 16 Inhibition of Vascular Endothelial Growth Factor (VEGF)Stimulated Proliferation of Endothelial Cell Growth (Assay 9)

[0963] The ability of various PRO polypeptides to inhibit VEGFstimulated proliferation of endothelial cells was tested. Polypeptidestesting positive in this assay are useful for inhibiting endothelialcell growth in mammals where such an effect would be beneficial, e.g.,for inhibiting tumor growth.

[0964] Specifically, bovine adrenal cortical capillary endothelial cells(ACE) (from primary culture, maximum of 12-14 passages) were plated in96-well plates at 500 cells/well per 100 microliter. Assay mediaincluded low glucose DMEM, 10% calf serum, 2 mM glutamine, and1×penicillin/streptomycin/fungizone. Control wells included thefollowing: (1) no ACE cells added; (2) ACE cells alone; (3) ACE cellsplus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGFplus 5 ng/ml LIF. The test samples, poly-his tagged PRO polypeptides (in100 microliter volumes), were then added to the wells (at dilutions of1%, 0.1% and 0.01%, respectively). The cell cultures were incubated for6-7 days at 37° C./5% CO₂. After the incubation, the media in the wellswas aspirated, and the cells were washed 1× with PBS. An acidphosphatase reaction mixture (100 microliter; 0.1M sodium acetate, pH5.5, 0.1% Triton X-100, 10 mM p-nitrophenyl phosphate) was then added toeach well. After a 2 hour incubation at 37° C., the reaction was stoppedby addition of 10 microliters 1N NaOH. Optical density (OD) was measuredon a microplate reader at 405 nm.

[0965] The activity of PRO polypeptides was calculated as the percentinhibition of VEGF (3 ng/ml) stimulated proliferation (as determined bymeasuring acid phosphatase activity at OD 405 run) relative to the cellswithout stimulation. TGF-beta was employed as an activity reference at 1ng/ml, since TGF-beta blocks 70-90% of VEGF-stimulated ACE cellproliferation. The results are indicative of the utility of the PROpolypeptides in cancer therapy and specifically in inhibiting tumorangiogenesis. Numerical values (relative inhibition) are determined bycalculating the percent inhibition of VEGF stimulated proliferation bythe PRO polypeptides relative to cells without stimulation and thendividing that percentage into the percent inhibition obtained by TGF-βat 1 ng/ml which is known to block 70-90% of VEGF stimulated cellproliferation. The results are considered positive if the PROpolypeptide exhibits 30% or greater inhibition of VEGF stimulation ofendothelial cell growth (relative inhibition 30% or greater).

[0966] PRO247, PRO720 and PRO4302 tested positive in this assay.

6.17. Example 17 Enhancement of Heart Neonatal Hypertrophy Induced byLIF+ET-1 (Assay 75)

[0967] This assay is designed to determine whether PRO polypeptides ofthe present invention show the ability to enhance neonatal hearthypertrophy induced by LIF and endothelin-1 (ET-1). A test compound thatprovides a positive response in the present assay would be useful forthe therapeutic treatment of cardiac insufficiency diseases or disorderscharacterized or associated with an undesired level of hypertrophy ofthe cardiac muscle.

[0968] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats (180μl at 7.5×10⁴/ml, serum <0.1, freshly isolated) are introduced on day 1to 96-well plates previously coated with DMEM/F12+4% FCS. Test PROpolypeptide samples or growth medium alone (negative control) are thenadded directly to the wells on day 2 in 20 μl volume. LIF+ET-1 are thenadded to the wells on day 3. The cells are stained after an additional 2days in culture and are then scored visually the next day. A positive inthe assay occurs when the PRO polypeptide treated myocytes obtain ascore greater than zero. A score of zero represents non-responsive cellswhereas scores of 1 or 2 represent enhancement (i.e. they are visuallylarger on the average or more numerous than the untreated myocytes).

[0969] PRO21 polypeptides tested positive in this assay.

6.18. Example 18 Detection of Endothelial Cell Apoptosis (FACS) (Assay96)

[0970] The ability of PRO polypeptides of the present invention toinduce apoptosis in endothelial cells was tested in human venousumbilical vein endothelial cells (HUVEC, Cell Systems) in gelatinizedT175 flasks using HUVEC cells below passage 10. PRO polypeptides testingpositive in this assay are expected to be useful for therapeuticallytreating conditions where apoptosis of endothelial cells would bebeneficial including, for example, the therapeutic treatment of tumors.

[0971] On day one, the cells were split [420,000 cells per gelatinized 6cm dishes—(11×10³cells/cm² Falcon, Primaria)] and grown in mediacontaining serum (CS-C, Cell System) overnight or for 16 hours to 24hours.

[0972] On day 2, the cells were washed 1× with 5 ml PBS; 3 ml of 0%serum medium was added with VEGF (100 ng/ml); and 30 μl of the PRO testcompound (final dilution 1%) or 0% serum medium (negative control) wasadded. The mixtures were incubated for 48 hours before harvesting.

[0973] The cells were then harvested for FACS analysis. The medium wasaspirated and the cells washed once with PBS. 5 ml of 1×trypsin wasadded to the cells in a T-175 flask, and the cells were allowed to standuntil they were released from the plate (about 5-10 minutes).Trypsinization was stopped by adding 5 ml of growth media. The cellswere spun at 1000 rpm for 5 minutes at 4° C. The media was aspirated andthe cells were resuspended in 10 ml of 10% serum complemented medium(Cell Systems), 5 μl of Annexin-FITC (BioVison) added and chilled tubeswere submitted for FACS. A positive result was determined to be enhancedapoptosis in the PRO polypeptide treated samples as compared to thenegative control.

[0974] PRO4302 polypeptide tested positive in this assay.

6.19. Example 19 Induction of c-fos in HUVEC Cells (Assay 123)

[0975] This assay is designed to determine whether PRO polypeptides showthe ability to induce c-fos in HUVEC cells. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of conditions or disorders where angiogenesiswould be beneficial including, for example, wound healing, and the like(as would agonists of these PRO polypeptides). Antagonists of the PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of cancerous tumors.

[0976] Human venous umbilical vein endothelial cells (HUVEC, CellSystems) in growth media (50% Ham's F12 w/o GHT: low glucose, and 50%DMEM without glycine: with NaHCO3, 1% glutamine, 10 mM HEPES, 10% FBS,10 ng/ml bFGF) were plated on 96-well microtiter plates at a celldensity of 5×10³cells/well. The day after plating (day 2), the cellswere starved for 24 hours by removing the growth media and replacingwith serum free media. On day 3, the cells are treated with 100 μl/welltest samples and controls (positive control=growth media; negativecontrol=Protein 32 buffer=10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol,pH 6.8). One plate of cells was incubated for 30 minutes at 37° C., in5% CO₂. Another plate of cells was incubated for 60 minutes at 37° C.,in 5% CO₂. The samples were removed, and RNA was harvested using theRNeasy 96 kit (Qiagen). Next, the RNA was assayed for c-fos, egr-1 andGAPDH induction using Taqman.

[0977] The measure of activity of the fold increase over the negativecontrol (Protein 32/HEPES buffer described above) value was by obtainedby calculating the fold increase of the ratio of c-fos to GAPDH in testsamples as compared to the negative control. The results are consideredpositive if the PRO polypeptide exhibits at least a two-fold value overthe negative buffer control.

[0978] PRO1376 polypeptide tested positive in this assay.

6.20. Example 20 Normal Human Iliac Artery Endothelial CellProliferation (Assay 138)

[0979] This assay is designed to determine whether PRO polypeptides ofthe present invention show the ability to modulate proliferation ofhuman iliac artery endothelial cells in culture and, therefore, functionas useful growth or inhibitory factors.

[0980] On day 0, human iliac artery endothelial cells (from cell lines,maximum of 12-14 passages) were plated in 96-well plates at 1000cells/well per 100 microliter and incubated overnight in complete media[epithelial cell growth media (EGM, Clonetics), plus supplements: humanepithelial growth factor (hEGF), bovine brain extract (BBE),hydrocortisone, GA-1000, and fetal bovine serum (FBS, Clonetics)]. Onday 1, complete media was replaced by basal media [EGM plus 1% FBS] andaddition of PRO polypeptides at 1%, 0.1% and 0.01% . On day 7, anassessment of cell proliferation was performed by Alamar Blue assayfollowed by Crystal Violet. Results are expressed as % of the cellgrowth observed with control buffer.

[0981] The following PRO polypeptides stimulated proliferation in thisassay: PRO214, PRO256, PRO363, PRO365, PRO791, PRO836, PRO1025, PRO1186,PRO1192, PRO1272, PRO1306, PRO1325, PRO1329, PRO1376, PRO1411, PRO1508,PRO1787, PRO1868, PRO4324, PRO4333, PRO4408, PRO4499, PRO9821, PRO9873,PRO10008, PRO10096, PRO19670, PRO20040, PRO20044 and PRO21384.

[0982] The following PRO polypeptides inhibited proliferation in thisassay: PRO238, PRO1029, PRO1274, PRO1279, PRO1419, PRO1890, PRO6006 andPRO28631.

6.21. Example 21 Pooled Human Umbilical Vein Endothelial CellProliferation (Assay 139)

[0983] This assay is designed to determine whether PRO polypeptides ofthe present invention show the ability to modulate proliferation ofpooled human umbilical vein endothelial cells in culture and, therefore,function as useful growth or inhibitory factors.

[0984] On day 0, pooled human umbilical vein endothelial cells (fromcell lines, maximum of 12-14 passages) were plated in 96-well plates at1000 cells/well per 100 microliter and incubated overnight in completemedia [epithelial cell growth media (EGM, Clonetics), plus supplements:human epithelial growth factor (hEGF), bovine brain extract (BBE),hydrocortisone, GA-1000, and fetal bovine serum (FBS, Clonetics)]. Onday 1, complete media was replaced by basal media [EGM plus 1% FBS] andaddition of PRO polypeptides at 1%, 0.1% and 0.01%. On day 7, anassessment of cell proliferation was performed by Alamar Blue assayfollowed by Crystal Violet. Results are expresses as % of the cellgrowth observed with control buffer.

[0985] The following PRO polypeptides stimulated proliferation in thisassay: PRO181, PRO205, PRO221, PRO231, PRO238, PRO241, PRO247, PRO256,PRO258, PRO263, PRO265, PRO295, PRO321, PRO322, PRO337, PRO363, PRO533,PRO697, PRO725, PRO771, PRO788, PRO819, PRO828, PRO846, PRO865, PRO1005,PRO1006, PRO1025, PRO1054, PRO1071, PRO1079, PRO1080, PRO1114, PRO1131,PRO1155, PRO1160, PRO1192, PRO1244, PRO1272, PRO1273, PRO1279, PRO1283,PRO1286, PRO1306, PRO1309, PRO1325, PRO1329, PRO1347, PRO1356, PRO1376,PRO1382, PRO1412, PRO1550, PRO1556, PRO1760, PRO1787, PRO1801, PRO1868,PRO1887, PRO3438, PRO3444, PRO4324, PRO4341, PRO4342, PRO4353, PRO4354,PRO4356, PRO4371, PRO4422, PRO4425, PRO5723, PRO5737, PRO6029, PRO6071,PRO10096 and PRO21055.

[0986] The following PRO polypeptides inhibited proliferation in thisassay: PRO229, PRO444, PRO827, PRO1007, PRO1075, PRO1184, PRO1190,PRO1195, PRO1419, PRO1474, PRO1477, PRO1488, PRO1782, PRO4302, PRO4405,PRO5725, PRO5776, PRO7436, PRO9771, PRO10008 and PRO21384.

6.22. Example 22 Human Coronary Artery Smooth Muscle Cell Proliferation(Assay 140)

[0987] This assay is designed to determine whether PRO polypeptides ofthe present invention show the ability to modulate proliferation ofhuman coronary artery smooth muscle cells in culture and, therefore,function as useful growth or inhibitory factors.

[0988] On day 0, human coronary artery smooth muscle cells (from celllines, maximum of 12-14 passages) were plated in 96-well plates at 1000cells/well per 100 microliter and incubated overnight in complete media[smooth muscle growth media (SmGM, Clonetics), plus supplements:insulin, human epithelial growth factor (hEGF), human fibroblast growthfactor (hFGF), GA-1000, and fetal bovine serum (FBS, Clonetics)]. On day1, complete media was replaced by basal media [SmGM plus 1% FBS] andaddition of PRO polypeptides at 1%, 0.1% and 0.01%. On day 7, anassessment of cell proliferation was performed by Alamar Blue assayfollowed by Crystal Violet. Results are expresses as % of the cellgrowth observed with control buffer.

[0989] The following PRO polypeptides stimulated proliferation in thisassay: PRO162, PRO182, PRO204, PRO221, PRO230, PRO256, PRO258, PRO533,PRO697, PRO725, PRO738, PRO826, PRO836, PRO840, PRO846, PRO865, PRO982,PRO1025, PRO1029, PRO1071, PRO1083, PRO1134, PRO1160, PRO1182, PRO1184,PRO1186, PRO1192, PRO1274, PRO1279, PRO1283, PRO1306, PRO1308, PRO1325,PRO1337, PRO1338, PRO1343, PRO1376, PRO1387, PRO1411, PRO1412, PRO1415,PRO1434, PRO1474, PRO1550, PRO1556, PRO1567, PRO1600, PRO1754, PRO1758,PRO1760, PRO1787, PRO1865, PRO1868, PRO1917, PRO1928, PRO3438, PRO3562,PRO4333, PRO4345, PRO4353, PRO4354, PRO4408, PRO4430, PRO4503, PRO6714,PRO9771, PRO9820, PRO9940, PRO10096, PRO21055, PRO21184 and PRO21366.

[0990] The following PRO polypeptides inhibited proliferation in thisassay: PRO181, PRO195, PRO1080, PRO1265, PRO1309, PRO1488, PRO4302,PRO4405 and PRO5725.

6.23. Example 23 Microarray Analysis to Detect Overexpression of PROPolypeptides in HUVEC Cells Treated with Growth Factors

[0991] This assay is designed to determine whether PRO polypeptides ofthe present invention show the ability to induce angiogenesis bystimulating endothelial cell tube formation in HUVEC cells.

[0992] Nucleic acid microarrays, often containing thousands of genesequences, are useful for identifying differentially expressed genes intissues exposed to various stimuli (e.g., growth factors) as compared totheir normal, unexposed counterparts. Using nucleic acid microarrays,test and control mRNA samples from test and control tissue samples arereverse transcribed and labeled to generate cDNA probes. The cDNA probesare then hybridized to an array of nucleic acids immobilized on a solidsupport. The array is configured such that the sequence and position ofeach member of the array is known. Hybridization of a labeled probe witha particular array member indicates that the sample from which the probewas derived expresses that gene. If the hybridization signal of a probefrom a test (exposed tissue) sample is greater than hybridization signalof a probe from a control (normal, unexposed tissue) sample, the gene orgenes overexpressed in the exposed tissue are identified. Theimplication of this result is that an overexpressed protein in anexposed tissue may be involved in the functional changes within thetissue following exposure to the stimuli (e.g., tube formation).

[0993] The methodology of hybridization of nucleic acids and microarraytechnology is well known in the art. In the present example, thespecific preparation of nucleic acids for hybridization and probes,slides, and hybridization conditions are all detailed in U.S.Provisional Patent Application Serial No. 60/193,767, filed on Mar. 31,2000 and which is herein incorporated by reference.

[0994] In the present example, HUVEC cells grown in either collagen gelsor fibrin gels were induced to form tubes by the addition of variousgrowth factors. Specifically, collagen gels were prepared as describedpreviously in Yang et al., American J. Pathology, 1999, 155(3):887-895and Xin et al., American J. Pathology, 2001, 158(3):1111-1120. Followinggelation of the HUVEC cells, 1×basal medium containing M199 supplementedwith 1% FBS, 1×ITS, 2 mM L-glutamine, 50 μg/ml ascorbic acid, 26.5 mMNaHCO₃, 100 U/ml penicillin and 100 U/ml streptomycin was added. Tubeformation was elicited by the inclusion in the culture media of either amixture of phorbol myrsitate acetate (50 nM), vascular endothelial cellgrowth factor (40 ng/ml) and basic fibroblast growth factor (40 ng/ml)(“PMA growth factor mix”) or hepatocyte growth factor (40 ng/ml) andvascular endothelial cell growth factor (40 ng/ml) (HGF/VEGF mix) forthe indicated period of time. Fibrin Gels were prepared by suspendingHuvec (4×10⁵ cells/ml) in M199 containing 1% fetal bovine serum(Hyclone) and human fibrinogen (2.5 mg/ml). Thrombin (50 U/ml) was thenadded to the fibrinogen suspension at a ratio of 1 part thrombinsolution:30 parts fibrinogen suspension. The solution was then layeredonto 10 cm tissue culture plates (total volume: 15 ml/plate) and allowedto solidify at 37° C. for 20 min. Tissue culture media (10 ml of BMcontaining PMA (50 nM), bFGF (40 ng/ml) and VEGF (40 ng/ml)) was thenadded and the cells incubated at 37° C. in 5% CO₂ in air for theindicated period of time.

[0995] Total RNA was extracted from the HUVEC cells incubated for 0, 4,8, 24, 40 and 50 hours in the different matrix and media combinationsusing a TRIzol extraction followed by a second purification usingRNAeasy Mini Kit (Qiagen). The total RNA was used to prepare cRNA whichwas then hybridized to the microarrays.

[0996] In the present experiments, nucleic acid probes derived from theherein described PRO polypeptide-encoding nucleic acid sequences wereused in the creation of the microarray and RNA from the HUVEC cellsdescribed above were used for the hybridization thereto. Pairwisecomparisons were made using time 0 chips as a baseline. Three replicatesamples were analyzed for each experimental condition and time. Hencethere were 3 time 0 samples for each treatment and 3 replicates of eachsuccessive time point. Therefore, a 3 by 3 comparison was performed foreach time point compared against each time 0 point. This resulted in 9comparisons per time point. Only those genes that had increasedexpression in all three non-time-0 replicates in each of the differentmatrix and media combinations as compared to any of the three time zeroreplicates were considered positive. Although this stringent method ofdata analysis does allow for false negatives, it minimizes falsepositives.

[0997] PRO178, PRO195, PRO228, PRO301, PRO302, PRO532, PRO724, PRO730,PRO734, PRO793, PRO871, PRO938, PRO1012, PRO1120, PRO1139, PRO1198,PRO1287, PRO1361, PRO1864, PRO1873, PRO2010, PRO3579, PRO4313, PRO4527,PRO4538, PRO4553, PRO4995, PRO5730, PRO6008, PRO7223,

[0998] PRO7248 and PRO7261 tested positive in this assay.

6.24. Example 24 In situ Hybridization

[0999] In situ hybridization is a powerful and versatile technique forthe detection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis, and aid in chromosome mapping.

[1000] In situ hybridization was performed following an optimizedversion of the protocol by Lu and Gillett, Cell Vision, 1: 169-176(1994), using PCR-generated ³³P-labeled riboprobes. Briefly,formalin-fixed, paraffin-embedded human tissues were sectioned,deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutesat 37° C., and further processed for in situ hybridization as describedby Lu and Gillett, supra. A (³³-P)UTP-labeled antisense riboprobe wasgenerated from a PCR product and hybridized at 55° C. overnight. Theslides were dipped in Kodak NTB2™ nuclear track emulsion and exposed for4 weeks.

[1001] 6.24.1. ³³P-Riboprobe Synthesis

[1002] 6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA <2000 Ci/mmol)were speed-vacuum dried. To each tube containing dried ³³P-UTP, thefollowing ingredients were added:

[1003] 2.0 μl 5×transcription buffer

[1004] 1.0 μl DTT (100 mM)

[1005] 2.0 μl NTP mix (2.5 mM: 10 μl each of 10 mM GTP, CTP & ATP+10 μlH₂O)

[1006] 1.0 μl UTP (50 μM)

[1007] 1.0 μl RNAsin

[1008] 1.0 μl DNA template (1 μg)

[1009] 1.0 μl H₂O

[1010] 1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

[1011] The tubes were incubated at 37° C. for one hour. A total of 1.0μl RQ1 DNase was added, followed by incubation at 37° C. for 15 minutes.A total of 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0and the mixturewas pipetted onto DE81 paper. The remaining solution was loaded in aMICROCON-50™ ultrafiltration unit, and spun using program 10 (6minutes). The filtration unit was inverted over a second tube and spunusing program 2 (3 minutes). After the final recovery spin, a total of100 μl TE was added, then 1 μl of the final product was pipetted on DE81paper and counted in 6 ml of BIOFLUOR II™.

[1012] The probe was run on a TBE/urea gel. A total of 1-3 μl of theprobe or 5 μl of RNA Mrk III was added to 3 μl of loading buffer. Afterheating on a 95° C. heat block for three minutes, the gel wasimmediately placed on ice. The wells of gel were flushed, and the samplewas loaded and run at 180-250 volts for 45 minutes. The gel was wrappedin plastic wrap (SARAN™ brand) and exposed to XAR film with anintensifying screen in a −70° C. freezer one hour overnight.

[1013] 6.24.2. ³³P-Hybridization

[1014] 6.24.2.1. Pretreatment of Frozen Sections

[1015] The slides were removed from the freezer, placed on aluminumtrays, and thawed at room temperature for 5 minutes. The trays wereplaced in a 55° C. incubator for five minutes to reduce condensation.The slides were fixed for 10 minutes in 4% paraformaldehyde on ice inthe fume hood, and washed in 0.5×SSC for 5 minutes, at room temperature(25 ml 20×SSC+975 ml SQ H₂O). After deproteination in 0.5 μg/mlproteinase K for 10 minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250ml prewarmed RNAse-free RNAse buffer), the sections were washed in0.5×SSC for 10 minutes at room temperature. The sections were dehydratedin 70%, 95%, and 100% ethanol, 2 minutes each.

[1016] 6.24.2.2. Pretreatment of Paraffin-embedded sections

[1017] The slides were deparaffinized, placed in SQ H₂O, and rinsedtwice in 2×SSC at room temperature, for 5 minutes each time. Thesections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/mlin 250 ml RNase-free RNase buffer; 37° C., 15 minutes) for human embryotissue, or 8×proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30minutes) for formalin tissues. Subsequent rinsing in 0.5×SSC anddehydration were performed as described above.

[1018] 6.24.2.3. Prehybridization

[1019] The slides were laid out in a plastic box lined with Box buffer(4×SSC, 50% formamide)—saturated filter paper. The tissue was coveredwith 50 μl of hybridization buffer (3.75 g dextran sulfate+6 ml SQ H₂O),vortexed, and heated in the microwave for 2 minutes with the caploosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC, and9 ml SQ H₂O were added, and the tissue was vortexed well and incubatedat 42° C. for 1-4 hours.

[1020] 6.24.2.4. Hybridization

[1021] 1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide wereheated at 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer was added per slide. After vortexing, 50 μl ³³P mixwas added to 50 μl prehybridization on the slide. The slides wereincubated overnight at 55° C.

[1022] 6.24.2.5. Washes

[1023] Washing was done for 2×10 minutes with 2×SSC, EDTA at roomtemperature (400 ml 20×SSC+16 ml 0.25 M EDTA, V_(f)=4L), followed byRNAseA treatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 mlRnase buffer=20 μg/ml), The slides were washed 2×10 minutes with 2×SSC,EDTA at room temperature. The stringency wash conditions were asfollows: 2 hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA,V_(f)=4L).

[1024] 6.24.2.6. Oligonucleotides

[1025] In situ analysis was performed on three of the DNA sequencesdisclosed herein. The primers used to generate the probes and/or theprobes employed for these analyses are as follows: DNA33100-p1: 5′GGATTC TAA TAC GAC TCA CTA TAG GGC CGG GTG GAG GTG GAA CAG AAA3′ (SEQ IDNO:375) DNA33100-p2: 5′CTA TGA AAT TAA CCC TCA CTA AAG GGA CAC AGA CAGAGC CCC ATA CGC3′ (SEQ ID NO:376) DNA34431-p1: 5′GGA TTC TAA TAC GAC TCACTA TAG GGC CAG GGA AAT CCG GAT GTC TC3′ (SEQ ID NO:377) DNA34431-p2:5′CTA TGA AAT TAA CCC TCA CTA AAG GGA GTA AGG GGA TGC CAC CGA GTA3′ (SEQID NO:378) DNA38268-p1: 5′GGA TTC TAA TAC GAC TCA CTA TAG GGC CAG CTACCC GCA GGA GGA GG3′ (SEQ ID NO:379) DNA38268-p2: 5′CTA TGA AAT TAA CCCTCA CTA AAG GGA TCC CAG GTG ATG AGG TCC AGA3′ (SEQ ID NO:380) DNA64908probe:5′CCATCTCGGAGACCTTTGTGCAGCGTGTATACCAGCCTTACCTCACCACTTGCGACGGACACAGAGCC(SEQ ID NO:381)TGCAGCACCTACCGAACCATCTACCGGACTGCCTATCGCCGTAGCCCTGGGGTGACTCCCGCAAGCCTCGCTATGCTTGCTGCCCTGGTTGGAAGAGGACCAGTGGGCTCCCTGGGGCTTGTGGAGCAGCAATATGCCAGCCTCCATGTGGGAATGGAGGGAGTTGCATCCGCCCAGGACACTGCCGCTGCCCTGTGGGATGGCAGGGAGATACTTGCCAGACAGATGTTGATGAATGCAGTACAGGAGAGGCCAGTTGTCCCCAGCGCTGTGTCAATACTGTGGGAAGTTACTGGTGCCAGGGATGGGAGGGACAAAGCCCATCTGCAGATGGGACGCGCTGCCTGTCTAAGGAGGGGCCCTCCCGGTGGCCCCAACCCCACAGCAGGAGTGGACAGCA3′

[1026] 6.24.2.7. Results

[1027] In situ analysis was performed and the results from theseanalyses are as follows:

[1028] 6.24.2.7.1. DNA33100-1159 (PRO229) (Scavenger-R/CD6 homologTNFmotif)

[1029] A specific positive signal was observed in mononuclear phagocytes(macrophages) of fetal and adult spleen, liver, lymph node and thymus.All other tissues were negative.

[1030] 6.24.2.7.2. DNA34431-1177 (PRO263) (CD44)

[1031] A specific positive signal was observed in human fetal tissuesand placenta over mononuclear cells, with strong expression inepithelial cells of the adrenal cortex. All adult tissues were negative.

[1032] 6.24.2.7.3. DNA38268-1188 (PRO295) (Integrin)

[1033] A specific positive signal was observed in human fetal ganglioncells, fetal neurons, adult adrenal medulla and adult neurons. All othertissues were negative.

[1034] 6.24.2.7.4. DNA64908-1163-1 (PRO1449)

[1035] A specific positive signal was observed in the developingvasculature (from E7-E11), in endothelial cells and in progenitors ofendothelial cells in wholemount in situ hybridizations of mouse embryos(FIG. 375). Specific expression was also observed in a subset of bloodvessels and epidermis from E12 onward. A mouse orthologue of PRO1449which has about 78% amino acid identity with PRO1449 was used as theprobe.

[1036] In normal adult tissues, expression was low to absent. Whenpresent, expression was confined to the vasculature (FIG. 376). FIG. 376further shows that highest expression in adult tissues was observedregionally in vessels running within the white matter of the brain.Elevated expression was also observed in vasculature of many inflamedand diseased tissues, including, but not limited to, tumor vasculature.

[1037] Following electroporation of the mouse orthologue of PRO1449 intothe choroid layer in the eyes of chicken embryos, new vessel formationwas observed in the electroporated eye (top right), but not in thecontrol side from the same embryo (top left), or an embryo that waselectroporated with a control cDNA (bottom right) (FIG. 377). cl 6.25.Example 25

Inhibition of Basic Fibroblast Growth Factor (bFGF) StimulatedProliferation of Endothelial Cell Growth

[1038] The ability of various PRO polypeptides to inhibit bFGFstimulated proliferation of endothelial cells was tested. Polypeptidestesting positive in this assay are useful for inhibiting endothelialcell growth in mammals where such an effect would be beneficial, e.g.,for inhibiting tumor growth.

[1039] Specifically, human venous umbilical vein endothelial cells(HUVEC, Cell Systems) in epithelial cell growth media (EGM, Clonetics)were plated on 96-well microtiter plates at a cell density of 5×10³cells/well in a volume of 100 μl/well. The day after plating (day 2),the cells were starved for 24 hours by removing the growth media andreplacing with starving media (M199 supplemented with 1% FBS, 2 mML-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin). On day 5,the cells are treated with either: (1) M199 with 10% FB M199 with 1%FBS; (3) M199 with 1% FBS and 20 ng/ml bFGF; (4) M199 with 1% FBS and 20ng/ml bFGF and PRO polypeptide (500 nM); (5) M199 with 1% FBS and 20ng/ml bFGF and PRO polypeptide (50 nM); or (6) M199 with 1% FBS and 20ng/ml bFGF and PRO polypeptide (5 nM). On day 8, an assessment of cellproliferation was performed by Alamar Blue assay. Optical density (OD)was measured on a microplate reader at excitation 530 and emission at590 nm.

[1040] The activity of PRO polypeptides was calculated as the percentinhibition of bFGF stimulated proliferation relative to the cellswithout stimulation. The results are indicative of the utility of thePRO polypeptides in cancer therapy and specifically in inhibiting tumorangiogenesis. Numerical values (relative inhibition) are determined bycalculating the percent inhibition of bFGF stimulated proliferation bythe PRO polypeptides relative to cells without stimulation. The resultsare considered positive if the PRO polypeptide exhibits 30% or greaterinhibition of bFEGF stimulation of endothelial cell growth.

[1041] PRO5725 tested positive in this assay.

[1042] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the construct(s)deposited, since the deposited embodiment(s) is/are intended as singleillustration(s) of certain aspects of the invention and any constructsthat are functionally equivalent are within the scope of this invention.The deposit of material(s) herein does not constitute an admission thatthe written description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20030100497). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

What is claimed is:
 1. An isolated nucleic acid molecule having at least80% nucleic acid sequence identity to a nucleotide sequence that encodesan amino acid sequence selected from the group consisting of the aminoacid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO: 4),FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ ID NO: 8), FIG. 10 (SEQ ID NO: 10),FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQ ID NO: 14), FIG. 16 (SEQ ID NO:16), FIG. 18 (SEQ ID NO: 18), FIG. 20 (SEQ ID NO: 20), FIG. 22 (SEQ IDNO: 22), FIG. 24 (SEQ ID NO: 24), FIG. 26 (SEQ ID NO: 26), FIG. 28 (SEQID NO: 28), FIG. 30 (SEQ ID NO: 30), FIG. 32 (SEQ ID NO: 32), FIG. 34(SEQ ID NO: 34), FIG. 36 (SEQ ID NO: 36), FIG. 38 (SEQ ID NO: 38), FIG.40 (SEQ ID NO: 40), FIG. 42 (SEQ ID NO: 42), FIG. 44 (SEQ ID NO: 44),FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQ ID NO: 48), FIG. 50 (SEQ ID NO:50), FIG. 52 (SEQ ID NO: 52), FIG. 54 (SEQ ID NO: 54), FIG. 56 (SEQ IDNO: 56), FIG. 58 (SEQ ID NO: 58), FIG. 60 (SEQ ID NO: 60), FIG. 62 (SEQID NO: 62), FIG. 64 (SEQ ID NO: 64), FIG. 66 (SEQ ID NO: 66), FIG. 68(SEQ ID NO: 68), FIG. 70 (SEQ ID NO: 70), FIG. 72 (SEQ ID NO: 72), FIG.74 (SEQ ID NO: 74), FIG. 76 (SEQ ID NO: 76), FIG. 78 (SEQ ID NO: 78),FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQ ID NO: 82), FIG. 84 (SEQ ID NO:84), FIG. 86 (SEQ ID NO: 86), FIG. 88 (SEQ ID NO: 88), FIG. 90 (SEQ IDNO: 90), FIG. 92 (SEQ ID NO: 92), FIG. 94 (SEQ ID NO: 94), FIG. 96 (SEQID NO: 96), FIG. 98 (SEQ ID NO: 98), FIG. 100 (SEQ ID NO: 100), FIG. 102(SEQ ID NO: 102), FIG. 104 (SEQ ID NO: 104), FIG. 106 (SEQ ID NO: 106),FIG. 108 (SEQ ID NO: 108), FIG. 110 (SEQ ID NO: 110), FIG. 112 (SEQ IDNO: 112), FIG. 114 (SEQ ID NO: 114), FIG. 116 (SEQ ID NO: 116), FIG. 118(SEQ ID NO: 118), FIG. 120 (SEQ ID NO: 120), FIG. 122 (SEQ ID NO: 122),FIG. 124 (SEQ ID NO: 124), FIG. 126 (SEQ ID NO: 126), FIG. 128 (SEQ IDNO: 128), FIG. 130 (SEQ ID NO: 130), FIG. 132 (SEQ ID NO: 132), FIG. 134(SEQ ID NO: 134), FIG. 136 (SEQ ID NO: 136), FIG. 138 (SEQ ID NO: 138),FIG. 140 (SEQ ID NO: 140), FIG. 142 (SEQ ID NO: 142), FIG. 144 (SEQ IDNO: 144), FIG. 146 (SEQ ID NO: 146), FIG. 148 (SEQ ID NO: 148), FIG. 150(SEQ ID NO: 150), FIG. 152 (SEQ ID NO: 152), FIG. 154 (SEQ ID NO: 154),FIG. 156 (SEQ ID NO: 156), FIG. 158 (SEQ ID NO: 158), FIG. 160 (SEQ IDNO: 160), FIG. 162 (SEQ ID NO: 162), FIG. 164 (SEQ ID NO: 164), FIG. 166(SEQ ID NO: 166), FIG. 168 (SEQ ID NO: 168), FIG. 170 (SEQ ID NO: 170),FIG. 172 (SEQ ID NO: 172), FIG. 174 (SEQ ID NO: 174), FIG. 176 (SEQ IDNO: 176), FIG. 178 (SEQ ID NO: 178), FIG. 180 (SEQ ID NO: 180), FIG. 182(SEQ ID NO: 182), FIG. 184 (SEQ ID NO: 184), FIG. 186 (SEQ ID NO: 186),FIG. 188 (SEQ ID NO: 188), FIG. 190 (SEQ ID NO: 190), FIG. 192 (SEQ IDNO: 192), FIG. 194 (SEQ ID NO: 194), FIG. 196 (SEQ ID NO: 196), FIG. 198(SEQ ID NO: 198), FIG. 200 (SEQ ID NO: 200), FIG. 202 (SEQ ID NO: 202),FIG. 204 (SEQ ID NO: 204), FIG. 206 (SEQ ID NO: 206), FIG. 208 (SEQ IDNO: 208), FIG. 210 (SEQ ID NO: 210), FIG. 212 (SEQ ID NO: 212), FIG. 214(SEQ ID NO: 214), FIG. 216 (SEQ ID NO: 216), FIG. 218 (SEQ ID NO: 218),FIG. 220 (SEQ ID NO: 220), FIG. 222 (SEQ ID NO: 222), FIG. 224 (SEQ IDNO: 224), FIG. 226 (SEQ ID NO: 226), FIG. 228 (SEQ ID NO: 228), FIG. 230(SEQ ID NO: 230), FIG. 232 (SEQ ID NO: 232), FIG. 234 (SEQ ID NO: 234),FIG. 236 (SEQ ID NO: 236), FIG. 238 (SEQ ID NO: 238), FIG. 240 (SEQ IDNO: 240), FIG. 242 (SEQ ID NO: 242), FIG. 244 (SEQ ID NO: 244), FIG. 246(SEQ ID NO: 246), FIG. 248 (SEQ ID NO: 248), FIG. 250 (SEQ ID NO: 250),FIG. 252 (SEQ ID NO: 252), FIG. 254 (SEQ ID NO: 254), FIG. 256 (SEQ IDNO: 256), FIG. 258 (SEQ ID NO: 258), FIG. 260 (SEQ ID NO: 260), FIG. 262(SEQ ID NO: 262), FIG. 264 (SEQ ID NO: 264), FIG. 266 (SEQ ID NO: 266),FIG. 268 (SEQ ID NO: 268), FIG. 270 (SEQ ID NO: 270), FIG. 272 (SEQ IDNO: 272), FIG. 274 (SEQ ID NO: 274), FIG. 276 (SEQ ID NO: 276), FIG. 278(SEQ ID NO: 278), FIG. 280 (SEQ ID NO: 280), FIG. 282 (SEQ ID NO: 282),FIG. 284 (SEQ ID NO: 284), FIG. 286 (SEQ ID NO: 286), FIG. 288 (SEQ IDNO: 288), FIG. 290 (SEQ ID NO: 290), FIG. 292 (SEQ ID NO: 292), FIG. 294(SEQ ID NO: 294), FIG. 296 (SEQ ID NO: 296), FIG. 298 (SEQ ID NO: 298),FIG. 300 (SEQ ID NO: 300), FIG. 302 (SEQ ID NO: 302), FIG. 304 (SEQ IDNO: 304), FIG. 306 (SEQ ID NO: 306), FIG. 308 (SEQ ID NO: 308), FIG. 310(SEQ ID NO: 310), FIG. 312 (SEQ ID NO: 312), FIG. 314 (SEQ ID NO: 314),FIG. 316 (SEQ ID NO: 316), FIG. 318 (SEQ ID NO: 318), FIG. 320 (SEQ IDNO: 320), FIG. 322 (SEQ ID NO: 322), FIG. 324 (SEQ ID NO: 324), FIG. 326(SEQ ID NO: 326), FIG. 328 (SEQ ID NO: 328), FIG. 330 (SEQ ID NO: 330),FIG. 332 (SEQ ID NO: 332), FIG. 334 (SEQ ID NO: 334), FIG. 336 (SEQ IDNO: 336), FIG. 338 (SEQ ID NO: 338), FIG. 340 (SEQ ID NO: 340), FIG. 342(SEQ ID NO: 342), FIG. 344 (SEQ ID NO: 344), FIG. 346 (SEQ ID NO: 346),FIG. 348 (SEQ ID NO: 348), FIG. 350 (SEQ ID NO: 350), FIG. 352 (SEQ IDNO: 352), FIG. 354 (SEQ ID NO: 354), FIG. 356 (SEQ ID NO: 356), FIG. 358(SEQ ID NO: 358), FIG. 360 (SEQ ID NO: 360), FIG. 362 (SEQ ID NO: 362),FIG. 364 (SEQ ID NO: 364), FIG. 366 (SEQ ID NO: 366), FIG. 368 (SEQ IDNO: 368), FIG. 370 (SEQ ID NO: 370), FIG. 372 (SEQ ID NO: 372) and FIG.374 (SEQ ID NO: 374).
 2. An isolated nucleic acid molecule having atleast 80% nucleic acid sequence identity to a nucleotide sequenceselected from the group consisting of the nucleotide sequence shown inFIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO: 3), FIG. 5 (SEQ ID NO: 5),FIG. 7 (SEQ ID NO: 7), FIG. 9 (SEQ ID NO: 9), FIG. 11 (SEQ ID NO: 11),FIG. 13 (SEQ ID NO: 13), FIG. 15 (SEQ ID NO: 15), FIG. 17 (SEQ ID NO:17), FIG. 19 (SEQ ID NO: 19), FIG. 21 (SEQ ID NO: 21), FIG. 23 (SEQ IDNO: 23), FIG. 25 (SEQ ID NO: 25), FIG. 27 (SEQ ID NO: 27), FIG. 29 (SEQID NO: 29), FIG. 31 (SEQ ID NO: 31), FIG. 33 (SEQ ID NO: 33), FIG. 35(SEQ ID NO: 35), FIG. 37 (SEQ ID NO: 37), FIG. 39 (SEQ ID NO: 39), FIG.41 (SEQ ID NO: 41), FIG. 43 (SEQ ID NO: 43), FIG. 45 (SEQ ID NO: 45),FIG. 47 (SEQ ID NO: 47), FIG. 49 (SEQ ID NO: 49), FIG. 51 (SEQ ID NO:51), FIG. 53 (SEQ ID NO: 53), FIG. 55 (SEQ ID NO: 55), FIG. 57 (SEQ IDNO: 57), FIG. 59 (SEQ ID NO: 59), FIG. 61 (SEQ ID NO: 61), FIG. 63 (SEQID NO: 63), FIG. 65 (SEQ ID NO: 65), FIG. 67 (SEQ ID NO: 67), FIG. 69(SEQ ID NO: 69), FIG. 71 (SEQ ID NO: 71), FIG. 73 (SEQ ID NO: 73), FIGS.75A-75B (SEQ ID NO: 75), FIG. 77 (SEQ ID NO: 77), FIG. 79 (SEQ ID NO:79), FIG. 81 (SEQ ID NO: 81), FIG. 83 (SEQ ID NO: 83), FIG. 85 (SEQ IDNO: 85), FIG. 87 (SEQ ID NO: 87), FIG. 89 (SEQ ID NO: 89), FIG. 91 (SEQID NO: 91), FIG. 93 (SEQ ID NO: 93), FIG. 95 (SEQ ID NO: 95), FIG. 97(SEQ ID NO: 97), FIG. 99 (SEQ ID NO: 99), FIG. 101 (SEQ ID NO: 101),FIG. 103 (SEQ ID NO: 103), FIG. 105 (SEQ ID NO: 105), FIG. 107 (SEQ IDNO: 107), FIG. 109 (SEQ ID NO: 109), FIG. 111 (SEQ ID NO: 111), FIG. 113(SEQ ID NO: 113), FIG. 115 (SEQ ID NO: 115), FIG. 117 (SEQ ID NO: 117),FIG. 119 (SEQ ID NO: 119), FIG. 121 (SEQ ID NO: 121), FIG. 123 (SEQ IDNO: 123), FIG. 125 (SEQ ID NO: 125), FIG. 127 (SEQ ID NO: 127), FIG. 129(SEQ ID NO: 129), FIG. 131 (SEQ ID NO: 131), FIG. 133 (SEQ ID NO: 133),FIG. 135 (SEQ ID NO: 135), FIG. 137 (SEQ ID NO: 137), FIG. 139 (SEQ IDNO: 139), FIG. 141 (SEQ ID NO: 141), FIG. 143 (SEQ ID NO: 143), FIG. 145(SEQ ID NO: 145), FIG. 147 (SEQ ID NO: 147), FIG. 149 (SEQ ID NO: 149),FIG. 151 (SEQ ID NO: 151), FIG. 153 (SEQ ID NO: 153), FIG. 155 (SEQ IDNO: 155), FIG. 157 (SEQ ID NO: 157), FIG. 159 (SEQ ID NO: 159), FIG. 161(SEQ ID NO: 161), FIG. 163 (SEQ ID NO: 163), FIG. 165 (SEQ ID NO: 165),FIG. 167 (SEQ ID NO: 167), FIG. 169 (SEQ ID NO: 169), FIG. 171 (SEQ IDNO: 171), FIG. 173 (SEQ ID NO: 173), FIG. 175 (SEQ ID NO: 175), FIG. 177(SEQ ID NO: 177), FIG. 179 (SEQ ID NO: 179), FIG. 181 (SEQ ID NO: 181),FIG. 183 (SEQ ID NO: 183), FIG. 185 (SEQ ID NO: 185), FIG. 187 (SEQ IDNO: 187), FIG. 189 (SEQ ID NO: 189), FIG. 191 (SEQ ID NO: 191), FIG. 193(SEQ ID NO: 193), FIG. 195 (SEQ ID NO: 195), FIG. 197 (SEQ ID NO: 197),FIG. 199 (SEQ ID NO: 199), FIG. 201 (SEQ ID NO: 201), FIG. 203 (SEQ IDNO: 203), FIG. 205 (SEQ ID NO: 205), FIG. 207 (SEQ ID NO: 207), FIG. 209(SEQ ID NO: 209), FIG. 211 (SEQ ID NO: 211), FIG. 213 (SEQ ID NO: 213),FIG. 215 (SEQ ID NO: 215), FIG. 217 (SEQ ID NO: 217), FIG. 219 (SEQ IDNO: 219), FIG. 221 (SEQ ID NO: 221), FIG. 223 (SEQ ID NO: 223), FIG. 225(SEQ ID NO: 225), FIG. 227 (SEQ ID NO: 227), FIG. 229 (SEQ ID NO: 229),FIG. 231 (SEQ ID NO: 231), FIG. 233 (SEQ ID NO: 233), FIG. 235 (SEQ IDNO: 235), FIG. 237 (SEQ ID NO: 237), FIG. 239 (SEQ ID NO: 239), FIG. 241(SEQ ID NO: 241), FIG. 243 (SEQ ID NO: 243), FIG. 245 (SEQ ID NO: 245),FIG. 247 (SEQ ID NO: 247), FIG. 249 (SEQ ID NO: 249), FIG. 251 (SEQ IDNO: 251), FIG. 253 (SEQ ID NO: 253), FIG. 255 (SEQ ID NO: 255), FIG. 257(SEQ ID NO: 257), FIG. 259 (SEQ ID NO: 259), FIG. 261 (SEQ ID NO: 261),FIG. 263 (SEQ ID NO: 263), FIG. 265 (SEQ ID NO: 265), FIG. 267 (SEQ IDNO: 267), FIG. 269 (SEQ ID NO: 269), FIG. 271 (SEQ ID NO: 271), FIG. 273(SEQ ID NO: 273), FIG. 275 (SEQ ID NO: 275), FIG. 277 (SEQ ID NO: 277),FIG. 279 (SEQ ID NO: 279), FIG. 281 (SEQ ID NO: 281), FIG. 283 (SEQ IDNO: 283), FIG. 285 (SEQ ID NO: 285), FIG. 287 (SEQ ID NO: 287), FIGS.289A-289B (SEQ ID NO: 289), FIG. 291 (SEQ ID NO: 291), FIG. 293 (SEQ IDNO: 293), FIG. 295 (SEQ ID NO: 295), FIG. 297 (SEQ ID NO: 297), FIG. 299(SEQ ID NO: 299), FIG. 301 (SEQ ID NO: 301), FIG. 303 (SEQ ID NO: 303),FIG. 305 (SEQ ID NO: 305), FIG. 307 (SEQ ID NO: 307), FIG. 309 (SEQ IDNO: 309), FIGS. 311A-311B (SEQ ID NO: 311), FIG. 313 (SEQ ID NO: 313),FIG. 315 (SEQ ID NO: 315), FIG. 317 (SEQ ID NO: 317), FIG. 319 (SEQ IDNO: 319), FIG. 321 (SEQ ID NO: 321), FIG. 323 (SEQ ID NO: 323), FIG. 325(SEQ ID NO: 325), FIG. 327 (SEQ ID NO: 327), FIG. 329 (SEQ ID NO: 329),FIG. 331 (SEQ ID NO: 331), FIG. 333 (SEQ ID NO: 333), FIG. 335 (SEQ IDNO: 335), FIG. 337 (SEQ ID NO: 337), FIG. 339 (SEQ ID NO: 339), FIG. 341(SEQ ID NO: 341), FIG. 343 (SEQ ID NO: 343), FIG. 345 (SEQ ID NO: 345),FIG. 347 (SEQ ID NO: 347), FIG. 349 (SEQ ID NO: 349), FIGS. 351A-351B(SEQ ID NO: 351), FIG. 353 (SEQ ID NO: 353), FIG. 355 (SEQ ID NO: 355),FIG. 357 (SEQ ID NO: 357), FIG. 359 (SEQ ID NO: 359), FIG. 361 (SEQ IDNO: 361), FIG. 363 (SEQ ID NO: 363), FIG. 365 (SEQ ID NO: 365), FIG. 367(SEQ ID NO: 367), FIG. 369 (SEQ ID NO: 369), FIG. 371 (SEQ ID NO: 371)and FIG. 373 (SEQ ID NO: 373).
 3. An isolated nucleic acid moleculehaving at least 80% nucleic acid sequence identity to a nucleotidesequence selected from the group consisting of the full-length codingsequence of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1), FIG.3 (SEQ ID NO: 3), FIG. 5 (SEQ ID NO: 5), FIG. 7 (SEQ ID NO: 7), FIG. 9(SEQ ID NO: 9), FIG. 11 (SEQ ID NO: 11), FIG. 13 (SEQ ID NO: 13), FIG.15 (SEQ ID NO: 15), FIG. 17 (SEQ ID NO: 17), FIG. 19 (SEQ ID NO: 19),FIG. 21 (SEQ ID NO: 21), FIG. 23 (SEQ ID NO: 23), FIG. 25 (SEQ ID NO:25), FIG. 27 (SEQ ID NO: 27), FIG. 29 (SEQ ID NO: 29), FIG. 31 (SEQ IDNO: 31), FIG. 33 (SEQ ID NO: 33), FIG. 35 (SEQ ID NO: 35), FIG. 37 (SEQID NO: 37), FIG. 39 (SEQ ID NO: 39), FIG. 41 (SEQ ID NO: 41), FIG. 43(SEQ ID NO: 43), FIG. 45 (SEQ ID NO: 45), FIG. 47 (SEQ ID NO: 47), FIG.49 (SEQ ID NO: 49), FIG. 51 (SEQ ID NO: 51), FIG. 53 (SEQ ID NO: 53),FIG. 55 (SEQ ID NO: 55), FIG. 57 (SEQ ID NO: 57), FIG. 59 (SEQ ID NO:59), FIG. 61 (SEQ ID NO: 61), FIG. 63 (SEQ ID NO: 63), FIG. 65 (SEQ IDNO: 65), FIG. 67 (SEQ ID NO: 67), FIG. 69 (SEQ ID NO: 69), FIG. 71 (SEQID NO: 71), FIG. 73 (SEQ ID NO: 73), FIGS. 75A-75B (SEQ ID NO: 75), FIG.77 (SEQ ID NO: 77), FIG. 79 (SEQ ID NO: 79), FIG. 81 (SEQ ID NO: 81),FIG. 83 (SEQ ID NO: 83), FIG. 85 (SEQ ID NO: 85), FIG. 87 (SEQ ID NO:87), FIG. 89 (SEQ ID NO: 89), FIG. 91 (SEQ ID NO: 91), FIG. 93 (SEQ IDNO: 93), FIG. 95 (SEQ ID NO: 95), FIG. 97 (SEQ ID NO: 97), FIG. 99 (SEQID NO: 99), FIG. 101 (SEQ ID NO: 101), FIG. 103 (SEQ ID NO: 103), FIG.105 (SEQ ID NO: 105), FIG. 107 (SEQ ID NO: 107), FIG. 109 (SEQ ID NO:109), FIG. 111 (SEQ ID NO: 111), FIG. 113 (SEQ ID NO: 113), FIG. 115(SEQ ID NO: 115), FIG. 117 (SEQ ID NO: 117), FIG. 119 (SEQ ID NO: 119),FIG. 121 (SEQ ID NO: 121), FIG. 123 (SEQ ID NO: 123), FIG. 125 (SEQ IDNO: 125), FIG. 127 (SEQ ID NO: 127), FIG. 129 (SEQ ID NO: 129), FIG. 131(SEQ ID NO: 131), FIG. 133 (SEQ ID NO: 133), FIG. 135 (SEQ ID NO: 135),FIG. 137 (SEQ ID NO: 137), FIG. 139 (SEQ ID NO: 139), FIG. 141 (SEQ IDNO: 141), FIG. 143 (SEQ ID NO: 143), FIG. 145 (SEQ ID NO: 145), FIG. 147(SEQ ID NO: 147), FIG. 149 (SEQ ID NO: 149), FIG. 151 (SEQ ID NO: 151),FIG. 153 (SEQ ID NO: 153), FIG. 155 (SEQ ID NO: 155), FIG. 157 (SEQ IDNO: 157), FIG. 159 (SEQ ID NO: 159), FIG. 161 (SEQ ID NO: 161), FIG. 163(SEQ ID NO: 163), FIG. 165 (SEQ ID NO: 165), FIG. 167 (SEQ ID NO: 167),FIG. 169 (SEQ ID NO: 169), FIG. 171 (SEQ ID NO: 171), FIG. 173 (SEQ IDNO: 173), FIG. 175 (SEQ ID NO: 175), FIG. 177 (SEQ ID NO: 177), FIG. 179(SEQ ID NO: 179), FIG. 181 (SEQ ID NO: 181), FIG. 183 (SEQ ID NO: 183),FIG. 185 (SEQ ID NO: 185), FIG. 187 (SEQ ID NO: 187), FIG. 189 (SEQ IDNO: 189), FIG. 191 (SEQ ID NO: 191), FIG. 193 (SEQ ID NO: 193), FIG. 195(SEQ ID NO: 195), FIG. 197 (SEQ ID NO: 197), FIG. 199 (SEQ ID NO: 199),FIG. 201 (SEQ ID NO: 201), FIG. 203 (SEQ ID NO: 203), FIG. 205 (SEQ IDNO: 205), FIG. 207 (SEQ ID NO: 207), FIG. 209 (SEQ ID NO: 209), FIG. 211(SEQ ID NO: 211), FIG. 213 (SEQ ID NO: 213), FIG. 215 (SEQ ID NO: 215),FIG. 217 (SEQ ID NO: 217), FIG. 219 (SEQ ID NO: 219), FIG. 221 (SEQ IDNO: 221), FIG. 223 (SEQ ID NO: 223), FIG. 225 (SEQ ID NO: 225), FIG. 227(SEQ ID NO: 227), FIG. 229 (SEQ ID NO: 229), FIG. 231 (SEQ ID NO: 231),FIG. 233 (SEQ ID NO: 233), FIG. 235 (SEQ ID NO: 235), FIG. 237 (SEQ IDNO: 237), FIG. 239 (SEQ ID NO: 239), FIG. 241 (SEQ ID NO: 241), FIG. 243(SEQ ID NO: 243), FIG. 245 (SEQ ID NO: 245), FIG. 247 (SEQ ID NO: 247),FIG. 249 (SEQ ID NO: 249), FIG. 251 (SEQ ID NO: 251), FIG. 253 (SEQ IDNO: 253), FIG. 255 (SEQ ID NO: 255), FIG. 257 (SEQ ID NO: 257), FIG. 259(SEQ ID NO: 259), FIG. 261 (SEQ ID NO: 261), FIG. 263 (SEQ ID NO: 263),FIG. 265 (SEQ ID NO: 265), FIG. 267 (SEQ ID NO: 267), FIG. 269 (SEQ IDNO: 269), FIG. 271 (SEQ ID NO: 271), FIG. 273 (SEQ ID NO: 273), FIG. 275(SEQ ID NO: 275), FIG. 277 (SEQ ID NO: 277), FIG. 279 (SEQ ID NO: 279),FIG. 281 (SEQ ID NO: 281), FIG. 283 (SEQ ID NO: 283), FIG. 285 (SEQ IDNO: 285), FIG. 287 (SEQ ID NO: 287), FIGS. 289A-289B (SEQ ID NO: 289),FIG. 291 (SEQ ID NO: 291), FIG. 293 (SEQ ID NO: 293), FIG. 295 (SEQ IDNO: 295), FIG. 297 (SEQ ID NO: 297), FIG. 299 (SEQ ID NO: 299), FIG. 301(SEQ ID NO: 301), FIG. 303 (SEQ ID NO: 303), FIG. 305 (SEQ ID NO: 305),FIG. 307 (SEQ ID NO: 307), FIG. 309 (SEQ ID NO: 309), FIGS. 311A-311B(SEQ ID NO: 311), FIG. 313 (SEQ ID NO: 313), FIG. 315 (SEQ ID NO: 315),FIG. 317 (SEQ ID NO: 317), FIG. 319 (SEQ ID NO: 319), FIG. 321 (SEQ IDNO: 321), FIG. 323 (SEQ ID NO: 323), FIG. 325 (SEQ ID NO: 325), FIG. 327(SEQ ID NO: 327), FIG. 329 (SEQ ID NO: 329), FIG. 331 (SEQ ID NO: 331),FIG. 333 (SEQ ID NO: 333), FIG. 335 (SEQ ID NO: 335), FIG. 337 (SEQ IDNO: 337), FIG. 339 (SEQ ID NO: 339), FIG. 341 (SEQ ID NO: 341), FIG. 343(SEQ ID NO: 343), FIG. 345 (SEQ ID NO: 345), FIG. 347 (SEQ ID NO: 347),FIG. 349 (SEQ ID NO: 349), FIGS. 351A-351B (SEQ ID NO: 351), FIG. 353(SEQ ID NO: 353), FIG. 355 (SEQ ID NO: 355), FIG. 357 (SEQ ID NO: 357),FIG. 359 (SEQ ID NO: 359), FIG. 361 (SEQ ID NO: 361), FIG. 363 (SEQ IDNO: 363), FIG. 365 (SEQ ID NO: 365), FIG. 367 (SEQ ID NO: 367), FIG. 369(SEQ ID NO: 369), FIG. 371 (SEQ ID NO: 371) and FIG. 373 (SEQ ID NO:373).
 4. An isolated nucleic acid molecule having at least 80% nucleicacid sequence identity to the full-length coding sequence of the DNAdeposited under any ATCC accession number shown in Table
 7. 5. A vectorcomprising the nucleic acid of claim
 1. 6. A host cell comprising thevector of claim
 5. 7. The host cell of claim 6, wherein said cell is aCHO cell.
 8. The host cell of claim 6, wherein said cell is an E. coli.9. The host cell of claim 6, wherein said cell is a yeast cell.
 10. Aprocess for producing a PRO polypeptide comprising culturing the hostcell of claim 6 under conditions suitable for expression of said PROpolypeptide and recovering said PRO polypeptide from the cell culture.11. An isolated polypeptide having at least 80% amino acid sequenceidentity to an amino acid sequence selected from the group consisting ofthe amino acid sequence shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ IDNO: 4), FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ ID NO: 8), FIG. 10 (SEQ IDNO: 10), FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQ ID NO: 14), FIG. 16 (SEQID NO: 16), FIG. 18 (SEQ ID NO: 18), FIG. 20 (SEQ ID NO: 20), FIG. 22(SEQ ID NO: 22), FIG. 24 (SEQ ID NO: 24), FIG. 26 (SEQ ID NO: 26), FIG.28 (SEQ ID NO: 28), FIG. 30 (SEQ ID NO: 30), FIG. 32 (SEQ ID NO: 32),FIG. 34 (SEQ ID NO: 34), FIG. 36 (SEQ ID NO: 36), FIG. 38 (SEQ ID NO:38), FIG. 40 (SEQ ID NO: 40), FIG. 42 (SEQ ID NO: 42), FIG. 44 (SEQ IDNO: 44), FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQ ID NO: 48), FIG. 50 (SEQID NO: 50), FIG. 52 (SEQ ID NO: 52), FIG. 54 (SEQ ID NO: 54), FIG. 56(SEQ ID NO: 56), FIG. 58 (SEQ ID NO: 58), FIG. 60 (SEQ ID NO: 60), FIG.62 (SEQ ID NO: 62), FIG. 64 (SEQ ID NO: 64), FIG. 66 (SEQ ID NO: 66),FIG. 68 (SEQ ID NO: 68), FIG. 70 (SEQ ID NO: 70), FIG. 72 (SEQ ID NO:72), FIG. 74 (SEQ ID NO: 74), FIG. 76 (SEQ ID NO: 76), FIG. 78 (SEQ IDNO: 78), FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQ ID NO: 82), FIG. 84 (SEQID NO: 84), FIG. 86 (SEQ ID NO: 86), FIG. 88 (SEQ ID NO: 88), FIG. 90(SEQ ID NO: 90), FIG. 92 (SEQ ID NO: 92), FIG. 94 (SEQ ID NO: 94), FIG.96 (SEQ ID NO: 96), FIG. 98 (SEQ ID NO: 98), FIG. 100 (SEQ ID NO: 100),FIG. 102 (SEQ ID NO: 102), FIG. 104 (SEQ ID NO: 104), FIG. 106 (SEQ IDNO: 106), FIG. 108 (SEQ ID NO: 108), FIG. 110 (SEQ ID NO: 110), FIG. 112(SEQ ID NO: 112), FIG. 114 (SEQ ID NO: 114), FIG. 116 (SEQ ID NO: 116),FIG. 118 (SEQ ID NO: 118), FIG. 120 (SEQ ID NO: 120), FIG. 122 (SEQ IDNO: 122), FIG. 124 (SEQ ID NO: 124), FIG. 126 (SEQ ID NO: 126), FIG. 128(SEQ ID NO: 128), FIG. 130 (SEQ ID NO: 130), FIG. 132 (SEQ ID NO: 132),FIG. 134 (SEQ ID NO: 134), FIG. 136 (SEQ ID NO: 136), FIG. 138 (SEQ IDNO: 138), FIG. 140 (SEQ ID NO: 140), FIG. 142 (SEQ ID NO: 142), FIG. 144(SEQ ID NO: 144), FIG. 146 (SEQ ID NO: 146), FIG. 148 (SEQ ID NO: 148),FIG. 150 (SEQ ID NO: 150), FIG. 152 (SEQ ID NO: 152), FIG. 154 (SEQ IDNO: 154), FIG. 156 (SEQ ID NO: 156), FIG. 158 (SEQ ID NO: 158), FIG. 160(SEQ ID NO: 160), FIG. 162 (SEQ ID NO: 162), FIG. 164 (SEQ ID NO: 164),FIG. 166 (SEQ ID NO: 166), FIG. 168 (SEQ ID NO: 168), FIG. 170 (SEQ IDNO: 170), FIG. 172 (SEQ ID NO: 172), FIG. 174 (SEQ ID NO: 174), FIG. 176(SEQ ID NO: 176), FIG. 178 (SEQ ID NO: 178), FIG. 180 (SEQ ID NO: 180),FIG. 182 (SEQ ID NO: 182), FIG. 184 (SEQ ID NO: 184), FIG. 186 (SEQ IDNO: 186), FIG. 188 (SEQ ID NO: 188), FIG. 190 (SEQ ID NO: 190), FIG. 192(SEQ ID NO: 192), FIG. 194 (SEQ ID NO: 194), FIG. 196 (SEQ ID NO: 196),FIG. 198 (SEQ ID NO: 198), FIG. 200 (SEQ ID NO: 200), FIG. 202 (SEQ IDNO: 202), FIG. 204 (SEQ ID NO: 204), FIG. 206 (SEQ ID NO: 206), FIG. 208(SEQ ID NO: 208), FIG. 210 (SEQ ID NO: 210), FIG. 212 (SEQ ID NO: 212),FIG. 214 (SEQ ID NO: 214), FIG. 216 (SEQ ID NO: 216), FIG. 218 (SEQ IDNO: 218), FIG. 220 (SEQ ID NO: 220), FIG. 222 (SEQ ID NO: 222), FIG. 224(SEQ ID NO: 224), FIG. 226 (SEQ ID NO: 226), FIG. 228 (SEQ ID NO: 228),FIG. 230 (SEQ ID NO: 230), FIG. 232 (SEQ ID NO: 232), FIG. 234 (SEQ IDNO: 234), FIG. 236 (SEQ ID NO: 236), FIG. 238 (SEQ ID NO: 238), FIG. 240(SEQ ID NO: 240), FIG. 242 (SEQ ID NO: 242), FIG. 244 (SEQ ID NO: 244),FIG. 246 (SEQ ID NO: 246), FIG. 248 (SEQ ID NO: 248), FIG. 250 (SEQ IDNO: 250), FIG. 252 (SEQ ID NO: 252), FIG. 254 (SEQ ID NO: 254), FIG. 256(SEQ ID NO: 256), FIG. 258 (SEQ ID NO: 258), FIG. 260 (SEQ ID NO: 260),FIG. 262 (SEQ ID NO: 262), FIG. 264 (SEQ ID NO: 264), FIG. 266 (SEQ IDNO: 266), FIG. 268 (SEQ ID NO: 268), FIG. 270 (SEQ ID NO: 270), FIG. 272(SEQ ID NO: 272), FIG. 274 (SEQ ID NO: 274), FIG. 276 (SEQ ID NO: 276),FIG. 278 (SEQ ID NO: 278), FIG. 280 (SEQ ID NO: 280), FIG. 282 (SEQ IDNO: 282), FIG. 284 (SEQ ID NO: 284), FIG. 286 (SEQ ID NO: 286), FIG. 288(SEQ ID NO: 288), FIG. 290 (SEQ ID NO: 290), FIG. 292 (SEQ ID NO: 292),FIG. 294 (SEQ ID NO: 294), FIG. 296 (SEQ ID NO: 296), FIG. 298 (SEQ IDNO: 298), FIG. 300 (SEQ ID NO: 300), FIG. 302 (SEQ ID NO: 302), FIG. 304(SEQ ID NO: 304), FIG. 306 (SEQ ID NO: 306), FIG. 308 (SEQ ID NO: 308),FIG. 310 (SEQ ID NO: 310), FIG. 312 (SEQ ID NO: 312), FIG. 314 (SEQ IDNO: 314), FIG. 316 (SEQ ID NO: 316), FIG. 318 (SEQ ID NO: 318), FIG. 320(SEQ ID NO: 320), FIG. 322 (SEQ ID NO: 322), FIG. 324 (SEQ ID NO: 324),FIG. 326 (SEQ ID NO: 326), FIG. 328 (SEQ ID NO: 328), FIG. 330 (SEQ IDNO: 330), FIG. 332 (SEQ ID NO: 332), FIG. 334 (SEQ ID NO: 334), FIG. 336(SEQ ID NO: 336), FIG. 338 (SEQ ID NO: 338), FIG. 340 (SEQ ID NO: 340),FIG. 342 (SEQ ID NO: 342), FIG. 344 (SEQ ID NO: 344), FIG. 346 (SEQ IDNO: 346), FIG. 348 (SEQ ID NO: 348), FIG. 350 (SEQ ID NO: 350), FIG. 352(SEQ ID NO: 352), FIG. 354 (SEQ ID NO: 354), FIG. 356 (SEQ ID NO: 356),FIG. 358 (SEQ ID NO: 358), FIG. 360 (SEQ ID NO: 360), FIG. 362 (SEQ IDNO: 362), FIG. 364 (SEQ ID NO: 364), FIG. 366 (SEQ ID NO: 366), FIG. 368(SEQ ID NO: 368), FIG. 370 (SEQ ID NO: 370), FIG. 372 (SEQ ID NO: 372)and FIG. 374 (SEQ ID NO: 374).
 12. An isolated polypeptide having atleast 80% amino acid sequence identity to an amino acid sequence encodedby the full-length coding sequence of the DNA deposited under any ATCCaccession number shown in Table
 7. 13. A chimeric molecule comprising apolypeptide according to claim 11 fused to a heterologous amino acidsequence.
 14. The chimeric molecule of claim 13, wherein saidheterologous amino acid sequence is an epitope tag sequence.
 15. Thechimeric molecule of claim 13, wherein said heterologous amino acidsequence is a Fc region of an immunoglobulin.
 16. An antibody whichspecifically binds to a polypeptide according to claim
 11. 17. Theantibody of claim 16, wherein said antibody is a monoclonal antibody, ahumanized antibody or a single-chain antibody.
 18. An isolated nucleicacid molecule having at least 80% nucleic acid sequence identity to: (a)a nucleotide sequence encoding the polypeptide shown in FIG. 2 (SEQ IDNO: 2), FIG. 4 (SEQ ID NO: 4), FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ ID NO:8), FIG. 10 (SEQ ID NO: 10), FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQ IDNO: 14), FIG. 16 (SEQ ID NO: 16), FIG. 18 (SEQ ID NO: 18), FIG. 20 (SEQID NO: 20), FIG. 22 (SEQ ID NO: 22), FIG. 24 (SEQ ID NO: 24), FIG. 26(SEQ ID NO: 26), FIG. 28 (SEQ ID NO: 28), FIG. 30 (SEQ ID NO: 30), FIG.32 (SEQ ID NO: 32), FIG. 34 (SEQ ID NO: 34), FIG. 36 (SEQ ID NO: 36),FIG. 38 (SEQ ID NO: 38), FIG. 40 (SEQ ID NO: 40), FIG. 42 (SEQ ID NO:42), FIG. 44 (SEQ ID NO: 44), FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQ IDNO: 48), FIG. 50 (SEQ ID NO: 50), FIG. 52 (SEQ ID NO: 52), FIG. 54 (SEQID NO: 54), FIG. 56 (SEQ ID NO: 56), FIG. 58 (SEQ ID NO: 58), FIG. 60(SEQ ID NO: 60), FIG. 62 (SEQ ID NO: 62), FIG. 64 (SEQ ID NO: 64), FIG.66 (SEQ ID NO: 66), FIG. 68 (SEQ ID NO: 68), FIG. 70 (SEQ ID NO: 70),FIG. 72 (SEQ ID NO: 72), FIG. 74 (SEQ ID NO: 74), FIG. 76 (SEQ ID NO:76), FIG. 78 (SEQ ID NO: 78), FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQ IDNO: 82), FIG. 84 (SEQ ID NO: 84), FIG. 86 (SEQ ID NO: 86), FIG. 88 (SEQID NO: 88), FIG. 90 (SEQ ID NO: 90), FIG. 92 (SEQ ID NO: 92), FIG. 94(SEQ ID NO: 94), FIG. 96 (SEQ ID NO: 96), FIG. 98 (SEQ ID NO: 98), FIG.100 (SEQ ID NO: 100), FIG. 102 (SEQ ID NO: 102), FIG. 104 (SEQ ID NO:104), FIG. 106 (SEQ ID NO: 106), FIG. 108 (SEQ ID NO: 108), FIG. 110(SEQ ID NO: 110), FIG. 112 (SEQ ID NO: 112), FIG. 114 (SEQ ID NO: 114),FIG. 116 (SEQ ID NO: 116), FIG. 118 (SEQ ID NO: 118), FIG. 120 (SEQ IDNO: 120), FIG. 122 (SEQ ID NO: 122), FIG. 124 (SEQ ID NO: 124), FIG. 126(SEQ ID NO: 126), FIG. 128 (SEQ ID NO: 128), FIG. 130 (SEQ ID NO: 130),FIG. 132 (SEQ ID NO: 132), FIG. 134 (SEQ ID NO: 134), FIG. 136 (SEQ IDNO: 136), FIG. 138 (SEQ ID NO: 138), FIG. 140 (SEQ ID NO: 140), FIG. 142(SEQ ID NO: 142), FIG. 144 (SEQ ID NO: 144), FIG. 146 (SEQ ID NO: 146),FIG. 148 (SEQ ID NO: 148), FIG. 150 (SEQ ID NO: 150), FIG. 152 (SEQ IDNO: 152), FIG. 154 (SEQ ID NO: 154), FIG. 156 (SEQ ID NO: 156), FIG. 158(SEQ ID NO: 158), FIG. 160 (SEQ ID NO: 160), FIG. 162 (SEQ ID NO: 162),FIG. 164 (SEQ ID NO: 164), FIG. 166 (SEQ ID NO: 166), FIG. 168 (SEQ IDNO: 168), FIG. 170 (SEQ ID NO: 170), FIG. 172 (SEQ ID NO: 172), FIG. 174(SEQ ID NO: 174), FIG. 176 (SEQ ID NO: 176), FIG. 178 (SEQ ID NO: 178),FIG. 180 (SEQ ID NO: 180), FIG. 182 (SEQ ID NO: 182), FIG. 184 (SEQ IDNO: 184), FIG. 186 (SEQ ID NO: 186), FIG. 188 (SEQ ID NO: 188), FIG. 190(SEQ ID NO: 190), FIG. 192 (SEQ ID NO: 192), FIG. 194 (SEQ ID NO: 194),FIG. 196 (SEQ ID NO: 196), FIG. 198 (SEQ ID NO: 198), FIG. 200 (SEQ IDNO: 200), FIG. 202 (SEQ ID NO: 202), FIG. 204 (SEQ ID NO: 204), FIG. 206(SEQ ID NO: 206), FIG. 208 (SEQ ID NO: 208), FIG. 210 (SEQ ID NO: 210),FIG. 212 (SEQ ID NO: 212), FIG. 214 (SEQ ID NO: 214), FIG. 216 (SEQ IDNO: 216), FIG. 218 (SEQ ID NO: 218), FIG. 220 (SEQ ID NO: 220), FIG. 222(SEQ ID NO: 222), FIG. 224 (SEQ ID NO: 224), FIG. 226 (SEQ ID NO: 226),FIG. 228 (SEQ ID NO: 228), FIG. 230 (SEQ ID NO: 230), FIG. 232 (SEQ IDNO: 232), FIG. 234 (SEQ ID NO: 234), FIG. 236 (SEQ ID NO: 236), FIG. 238(SEQ ID NO: 238), FIG. 240 (SEQ ID NO: 240), FIG. 242 (SEQ ID NO: 242),FIG. 244 (SEQ ID NO: 244), FIG. 246 (SEQ ID NO: 246), FIG. 248 (SEQ IDNO: 248), FIG. 250 (SEQ ID NO: 250), FIG. 252 (SEQ ID NO: 252), FIG. 254(SEQ ID NO: 254), FIG. 256 (SEQ ID NO: 256), FIG. 258 (SEQ ID NO: 258),FIG. 260 (SEQ ID NO: 260), FIG. 262 (SEQ ID NO: 262), FIG. 264 (SEQ IDNO: 264), FIG. 266 (SEQ ID NO: 266), FIG. 268 (SEQ ID NO: 268), FIG. 270(SEQ ID NO: 270), FIG. 272 (SEQ ID NO: 272), FIG. 274 (SEQ ID NO: 274),FIG. 276 (SEQ ID NO: 276), FIG. 278 (SEQ ID NO: 278), FIG. 280 (SEQ IDNO: 280), FIG. 282 (SEQ ID NO: 282), FIG. 284 (SEQ ID NO: 284), FIG. 286(SEQ ID NO: 286), FIG. 288 (SEQ ID NO: 288), FIG. 290 (SEQ ID NO: 290),FIG. 292 (SEQ ID NO: 292), FIG. 294 (SEQ ID NO: 294), FIG. 296 (SEQ IDNO: 296), FIG. 298 (SEQ ID NO: 298), FIG. 300 (SEQ ID NO: 300), FIG. 302(SEQ ID NO: 302), FIG. 304 (SEQ ID NO: 304), FIG. 306 (SEQ ID NO: 306),FIG. 308 (SEQ ID NO: 308), FIG. 310 (SEQ ID NO: 310), FIG. 312 (SEQ IDNO: 312), FIG. 314 (SEQ ID NO: 314), FIG. 316 (SEQ ID NO: 316), FIG. 318(SEQ ID NO: 318), FIG. 320 (SEQ ID NO: 320), FIG. 322 (SEQ ID NO: 322),FIG. 324 (SEQ ID NO: 324), FIG. 326 (SEQ ID NO: 326), FIG. 328 (SEQ IDNO: 328), FIG. 330 (SEQ ID NO: 330), FIG. 332 (SEQ ID NO: 332), FIG. 334(SEQ ID NO: 334), FIG. 336 (SEQ ID NO: 336), FIG. 338 (SEQ ID NO: 338),FIG. 340 (SEQ ID NO: 340), FIG. 342 (SEQ ID NO: 342), FIG. 344 (SEQ IDNO: 344), FIG. 346 (SEQ ID NO: 346), FIG. 348 (SEQ ID NO: 348), FIG. 350(SEQ ID NO: 350), FIG. 352 (SEQ ID NO: 352), FIG. 354 (SEQ ID NO: 354),FIG. 356 (SEQ ID NO: 356), FIG. 358 (SEQ ID NO: 358), FIG. 360 (SEQ IDNO: 360), FIG. 362 (SEQ ID NO: 362), FIG. 364 (SEQ ID NO: 364), FIG. 366(SEQ ID NO: 366), FIG. 368 (SEQ ID NO: 368), FIG. 370 (SEQ ID NO: 370),FIG. 372 (SEQ ID NO: 372) or FIG. 374 (SEQ ID NO: 374), lacking itsassociated signal peptide; (b) a nucleotide sequence encoding anextracellular domain of the polypeptide shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4), FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ ID NO: 8),FIG. 10 (SEQ ID NO: 10), FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQ ID NO:14), FIG. 16 (SEQ ID NO: 16), FIG. 18 (SEQ ID NO: 18), FIG. 20 (SEQ IDNO: 20), FIG. 22 (SEQ ID NO: 22), FIG. 24 (SEQ ID NO: 24), FIG. 26 (SEQID NO: 26), FIG. 28 (SEQ ID NO: 28), FIG. 30 (SEQ ID NO: 30), FIG. 32(SEQ ID NO: 32), FIG. 34 (SEQ ID NO: 34), FIG. 36 (SEQ ID NO: 36), FIG.38 (SEQ ID NO: 38), FIG. 40 (SEQ ID NO: 40), FIG. 42 (SEQ ID NO: 42),FIG. 44 (SEQ ID NO: 44), FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQ ID NO:48), FIG. 50 (SEQ ID NO: 50), FIG. 52 (SEQ ID NO: 52), FIG. 54 (SEQ IDNO: 54), FIG. 56 (SEQ ID NO: 56), FIG. 58 (SEQ ID NO: 58), FIG. 60 (SEQID NO: 60), FIG. 62 (SEQ ID NO: 62), FIG. 64 (SEQ ID NO: 64), FIG. 66(SEQ ID NO: 66), FIG. 68 (SEQ ID NO: 68), FIG. 70 (SEQ ID NO: 70), FIG.72 (SEQ ID NO: 72), FIG. 74 (SEQ ID NO: 74), FIG. 76 (SEQ ID NO: 76),FIG. 78 (SEQ ID NO: 78), FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQ ID NO:82), FIG. 84 (SEQ ID NO: 84), FIG. 86 (SEQ ID NO: 86), FIG. 88 (SEQ IDNO: 88), FIG. 90 (SEQ ID NO: 90), FIG. 92 (SEQ ID NO: 92), FIG. 94 (SEQID NO: 94), FIG. 96 (SEQ ID NO: 96), FIG. 98 (SEQ ID NO: 98), FIG. 100(SEQ ID NO: 100), FIG. 102 (SEQ ID NO: 102), FIG. 104 (SEQ ID NO: 104),FIG. 106 (SEQ ID NO: 106), FIG. 108 (SEQ ID NO: 108), FIG. 110 (SEQ IDNO: 110), FIG. 112 (SEQ ID NO: 112), FIG. 114 (SEQ ID NO: 114), FIG. 116(SEQ ID NO: 116), FIG. 118 (SEQ ID NO: 118), FIG. 120 (SEQ ID NO: 120),FIG. 122 (SEQ ID NO: 122), FIG. 124 (SEQ ID NO: 124), FIG. 126 (SEQ IDNO: 126), FIG. 128 (SEQ ID NO: 128), FIG. 130 (SEQ ID NO: 130), FIG. 132(SEQ ID NO: 132), FIG. 134 (SEQ ID NO: 134), FIG. 136 (SEQ ID NO: 136),FIG. 138 (SEQ ID NO: 138), FIG. 140 (SEQ ID NO: 140), FIG. 142 (SEQ IDNO: 142), FIG. 144 (SEQ ID NO: 144), FIG. 146 (SEQ ID NO: 146), FIG. 148(SEQ ID NO: 148), FIG. 150 (SEQ ID NO: 150), FIG. 152 (SEQ ID NO: 152),FIG. 154 (SEQ ID NO: 154), FIG. 156 (SEQ ID NO: 156), FIG. 158 (SEQ IDNO: 158), FIG. 160 (SEQ ID NO: 160), FIG. 162 (SEQ ID NO: 162), FIG. 164(SEQ ID NO: 164), FIG. 166 (SEQ ID NO: 166), FIG. 168 (SEQ ID NO: 168),FIG. 170 (SEQ ID NO: 170), FIG. 172 (SEQ ID NO: 172), FIG. 174 (SEQ IDNO: 174), FIG. 176 (SEQ ID NO: 176), FIG. 178 (SEQ ID NO: 178), FIG. 180(SEQ ID NO: 180), FIG. 182 (SEQ ID NO: 182), FIG. 184 (SEQ ID NO: 184),FIG. 186 (SEQ ID NO: 186), FIG. 188 (SEQ ID NO: 188), FIG. 190 (SEQ IDNO: 190), FIG. 192 (SEQ ID NO: 192), FIG. 194 (SEQ ID NO: 194), FIG. 196(SEQ ID NO: 196), FIG. 198 (SEQ ID NO: 198), FIG. 200 (SEQ ID NO: 200),FIG. 202 (SEQ ID NO: 202), FIG. 204 (SEQ ID NO: 204), FIG. 206 (SEQ IDNO: 206), FIG. 208 (SEQ ID NO: 208), FIG. 210 (SEQ ID NO: 210), FIG. 212(SEQ ID NO: 212), FIG. 214 (SEQ ID NO: 214), FIG. 216 (SEQ ID NO: 216),FIG. 218 (SEQ ID NO: 218), FIG. 220 (SEQ ID NO: 220), FIG. 222 (SEQ IDNO: 222), FIG. 224 (SEQ ID NO: 224), FIG. 226 (SEQ ID NO: 226), FIG. 228(SEQ ID NO: 228), FIG. 230 (SEQ ID NO: 230), FIG. 232 (SEQ ID NO: 232),FIG. 234 (SEQ ID NO: 234), FIG. 236 (SEQ ID NO: 236), FIG. 238 (SEQ IDNO: 238), FIG. 240 (SEQ ID NO: 240), FIG. 242 (SEQ ID NO: 242), FIG. 244(SEQ ID NO: 244), FIG. 246 (SEQ ID NO: 246), FIG. 248 (SEQ ID NO: 248),FIG. 250 (SEQ ID NO: 250), FIG. 252 (SEQ ID NO: 252), FIG. 254 (SEQ IDNO: 254), FIG. 256 (SEQ ID NO: 256), FIG. 258 (SEQ ID NO: 258), FIG. 260(SEQ ID NO: 260), FIG. 262 (SEQ ID NO: 262), FIG. 264 (SEQ ID NO: 264),FIG. 266 (SEQ ID NO: 266), FIG. 268 (SEQ ID NO: 268), FIG. 270 (SEQ IDNO: 270), FIG. 272 (SEQ ID NO: 272), FIG. 274 (SEQ ID NO: 274), FIG. 276(SEQ ID NO: 276), FIG. 278 (SEQ ID NO: 278), FIG. 280 (SEQ ID NO: 280),FIG. 282 (SEQ ID NO: 282), FIG. 284 (SEQ ID NO: 284), FIG. 286 (SEQ IDNO: 286), FIG. 288 (SEQ ID NO: 288), FIG. 290 (SEQ ID NO: 290), FIG. 292(SEQ ID NO: 292), FIG. 294 (SEQ ID NO: 294), FIG. 296 (SEQ ID NO: 296),FIG. 298 (SEQ ID NO: 298), FIG. 300 (SEQ ID NO: 300), FIG. 302 (SEQ IDNO: 302), FIG. 304 (SEQ ID NO: 304), FIG. 306 (SEQ ID NO: 306), FIG. 308(SEQ ID NO: 308), FIG. 310 (SEQ ID NO: 310), FIG. 312 (SEQ ID NO: 312),FIG. 314 (SEQ ID NO: 314), FIG. 316 (SEQ ID NO: 316), FIG. 318 (SEQ IDNO: 318), FIG. 320 (SEQ ID NO: 320), FIG. 322 (SEQ ID NO: 322), FIG. 324(SEQ ID NO: 324), FIG. 326 (SEQ ID NO: 326), FIG. 328 (SEQ ID NO: 328),FIG. 330 (SEQ ID NO: 330), FIG. 332 (SEQ ID NO: 332), FIG. 334 (SEQ IDNO: 334), FIG. 336 (SEQ ID NO: 336), FIG. 338 (SEQ ID NO: 338), FIG. 340(SEQ ID NO: 340), FIG. 342 (SEQ ID NO: 342), FIG. 344 (SEQ ID NO: 344),FIG. 346 (SEQ ID NO: 346), FIG. 348 (SEQ ID NO: 348), FIG. 350 (SEQ IDNO: 350), FIG. 352 (SEQ ID NO: 352), FIG. 354 (SEQ ID NO: 354), FIG. 356(SEQ ID NO: 356), FIG. 358 (SEQ ID NO: 358), FIG. 360 (SEQ ID NO: 360),FIG. 362 (SEQ ID NO: 362), FIG. 364 (SEQ ID NO: 364), FIG. 366 (SEQ IDNO: 366), FIG. 368 (SEQ ID NO: 368), FIG. 370 (SEQ ID NO: 370), FIG. 372(SEQ ID NO: 372) or FIG. 374 (SEQ ID NO: 374), with its associatedsignal peptide; or (c) a nucleotide sequence encoding an extracellulardomain of the polypeptide shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ IDNO: 4), FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ ID NO: 8), FIG. 10 (SEQ IDNO: 10), FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQ ID NO: 14), FIG. 16 (SEQID NO: 16), FIG. 18 (SEQ ID NO: 18), FIG. 20 (SEQ ID NO: 20), FIG. 22(SEQ ID NO: 22), FIG. 24 (SEQ ID NO: 24), FIG. 26 (SEQ ID NO: 26), FIG.28 (SEQ ID NO: 28), FIG. 30 (SEQ ID NO: 30), FIG. 32 (SEQ ID NO: 32),FIG. 34 (SEQ ID NO: 34), FIG. 36 (SEQ ID NO: 36), FIG. 38 (SEQ ID NO:38), FIG. 40 (SEQ ID NO: 40), FIG. 42 (SEQ ID NO: 42), FIG. 44 (SEQ IDNO: 44), FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQ ID NO: 48), FIG. 50 (SEQID NO: 50), FIG. 52 (SEQ ID NO: 52), FIG. 54 (SEQ ID NO: 54), FIG. 56(SEQ ID NO: 56), FIG. 58 (SEQ ID NO: 58), FIG. 60 (SEQ ID NO: 60), FIG.62 (SEQ ID NO: 62), FIG. 64 (SEQ ID NO: 64), FIG. 66 (SEQ ID NO: 66),FIG. 68 (SEQ ID NO: 68), FIG. 70 (SEQ ID NO: 70), FIG. 72 (SEQ ID NO:72), FIG. 74 (SEQ ID NO: 74), FIG. 76 (SEQ ID NO: 76), FIG. 78 (SEQ IDNO: 78), FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQ ID NO: 82), FIG. 84 (SEQID NO: 84), FIG. 86 (SEQ ID NO: 86), FIG. 88 (SEQ ID NO: 88), FIG. 90(SEQ ID NO: 90), FIG. 92 (SEQ ID NO: 92), FIG. 94 (SEQ ID NO: 94), FIG.96 (SEQ ID NO: 96), FIG. 98 (SEQ ID NO: 98), FIG. 100 (SEQ ID NO: 100),FIG. 102 (SEQ ID NO: 102), FIG. 104 (SEQ ID NO: 104), FIG. 106 (SEQ IDNO: 106), FIG. 108 (SEQ ID NO: 108), FIG. 110 (SEQ ID NO: 110), FIG. 112(SEQ ID NO: 112), FIG. 114 (SEQ ID NO: 114), FIG. 116 (SEQ ID NO: 116),FIG. 118 (SEQ ID NO: 118), FIG. 120 (SEQ ID NO: 120), FIG. 122 (SEQ IDNO: 122), FIG. 124 (SEQ ID NO: 124), FIG. 126 (SEQ ID NO: 126), FIG. 128(SEQ ID NO: 128), FIG. 130 (SEQ ID NO: 130), FIG. 132 (SEQ ID NO: 132),FIG. 134 (SEQ ID NO: 134), FIG. 136 (SEQ ID NO: 136), FIG. 138 (SEQ IDNO: 138), FIG. 140 (SEQ ID NO: 140), FIG. 142 (SEQ ID NO: 142), FIG. 144(SEQ ID NO: 144), FIG. 146 (SEQ ID NO: 146), FIG. 148 (SEQ ID NO: 148),FIG. 150 (SEQ ID NO: 150), FIG. 152 (SEQ ID NO: 152), FIG. 154 (SEQ IDNO: 154), FIG. 156 (SEQ ID NO: 156), FIG. 158 (SEQ ID NO: 158), FIG. 160(SEQ ID NO: 160), FIG. 162 (SEQ ID NO: 162), FIG. 164 (SEQ ID NO: 164),FIG. 166 (SEQ ID NO: 166), FIG. 168 (SEQ ID NO: 168), FIG. 170 (SEQ IDNO: 170), FIG. 172 (SEQ ID NO: 172), FIG. 174 (SEQ ID NO: 174), FIG. 176(SEQ ID NO: 176), FIG. 178 (SEQ ID NO: 178), FIG. 180 (SEQ ID NO: 180),FIG. 182 (SEQ ID NO: 182), FIG. 184 (SEQ ID NO: 184), FIG. 186 (SEQ IDNO: 186), FIG. 188 (SEQ ID NO: 188), FIG. 190 (SEQ ID NO: 190), FIG. 192(SEQ ID NO: 192), FIG. 194 (SEQ ID NO: 194), FIG. 196 (SEQ ID NO: 196),FIG. 198 (SEQ ID NO: 198), FIG. 200 (SEQ ID NO: 200), FIG. 202 (SEQ IDNO: 202), FIG. 204 (SEQ ID NO: 204), FIG. 206 (SEQ ID NO: 206), FIG. 208(SEQ ID NO: 208), FIG. 210 (SEQ ID NO: 210), FIG. 212 (SEQ ID NO: 212),FIG. 214 (SEQ ID NO: 214), FIG. 216 (SEQ ID NO: 216), FIG. 218 (SEQ IDNO: 218), FIG. 220 (SEQ ID NO: 220), FIG. 222 (SEQ ID NO: 222), FIG. 224(SEQ ID NO: 224), FIG. 226 (SEQ ID NO: 226), FIG. 228 (SEQ ID NO: 228),FIG. 230 (SEQ ID NO: 230), FIG. 232 (SEQ ID NO: 232), FIG. 234 (SEQ IDNO: 234), FIG. 236 (SEQ ID NO: 236), FIG. 238 (SEQ ID NO: 238), FIG. 240(SEQ ID NO: 240), FIG. 242 (SEQ ID NO: 242), FIG. 244 (SEQ ID NO: 244),FIG. 246 (SEQ ID NO: 246), FIG. 248 (SEQ ID NO: 248), FIG. 250 (SEQ IDNO: 250), FIG. 252 (SEQ ID NO: 252), FIG. 254 (SEQ ID NO: 254), FIG. 256(SEQ ID NO: 256), FIG. 258 (SEQ ID NO: 258), FIG. 260 (SEQ ID NO: 260),FIG. 262 (SEQ ID NO: 262), FIG. 264 (SEQ ID NO: 264), FIG. 266 (SEQ IDNO: 266), FIG. 268 (SEQ ID NO: 268), FIG. 270 (SEQ ID NO: 270), FIG. 272(SEQ ID NO: 272), FIG. 274 (SEQ ID NO: 274), FIG. 276 (SEQ ID NO: 276),FIG. 278 (SEQ ID NO: 278), FIG. 280 (SEQ ID NO: 280), FIG. 282 (SEQ IDNO: 282), FIG. 284 (SEQ ID NO: 284), FIG. 286 (SEQ ID NO: 286), FIG. 288(SEQ ID NO: 288), FIG. 290 (SEQ ID NO: 290), FIG. 292 (SEQ ID NO: 292),FIG. 294 (SEQ ID NO: 294), FIG. 296 (SEQ ID NO: 296), FIG. 298 (SEQ IDNO: 298), FIG. 300 (SEQ ID NO: 300), FIG. 302 (SEQ ID NO: 302), FIG. 304(SEQ ID NO: 304), FIG. 306 (SEQ ID NO: 306), FIG. 308 (SEQ ID NO: 308),FIG. 310 (SEQ ID NO: 310), FIG. 312 (SEQ ID NO: 312), FIG. 314 (SEQ IDNO: 314), FIG. 316 (SEQ ID NO: 316), FIG. 318 (SEQ ID NO: 318), FIG. 320(SEQ ID NO: 320), FIG. 322 (SEQ ID NO: 322), FIG. 324 (SEQ ID NO: 324),FIG. 326 (SEQ ID NO: 326), FIG. 328 (SEQ ID NO: 328), FIG. 330 (SEQ IDNO: 330), FIG. 332 (SEQ ID NO: 332), FIG. 334 (SEQ ID NO: 334), FIG. 336(SEQ ID NO: 336), FIG. 338 (SEQ ID NO: 338), FIG. 340 (SEQ ID NO: 340),FIG. 342 (SEQ ID NO: 342), FIG. 344 (SEQ ID NO: 344), FIG. 346 (SEQ IDNO: 346), FIG. 348 (SEQ ID NO: 348), FIG. 350 (SEQ ID NO: 350), FIG. 352(SEQ ID NO: 352), FIG. 354 (SEQ ID NO: 354), FIG. 356 (SEQ ID NO: 356),FIG. 358 (SEQ ID NO: 358), FIG. 360 (SEQ ID NO: 360), FIG. 362 (SEQ IDNO: 362), FIG. 364 (SEQ ID NO: 364), FIG. 366 (SEQ ID NO: 366), FIG. 368(SEQ ID NO: 368), FIG. 370 (SEQ ID NO: 370), FIG. 372 (SEQ ID NO: 372)or FIG. 374 (SEQ ID NO: 374), lacking its associated signal peptide. 19.An isolated polypeptide having at least 80% amino acid sequence identityto: (a) an amino acid sequence of the polypeptide shown in FIG. 2 (SEQID NO: 2), FIG. 4 (SEQ ID NO: 4), FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ IDNO: 8), FIG. 10 (SEQ ID NO: 10), FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQID NO: 14), FIG. 16 (SEQ ID NO: 16), FIG. 18 (SEQ ID NO: 18), FIG. 20(SEQ ID NO: 20), FIG. 22 (SEQ ID NO: 22), FIG. 24 (SEQ ID NO: 24), FIG.26 (SEQ ID NO: 26), FIG. 28 (SEQ ID NO: 28), FIG. 30 (SEQ ID NO: 30),FIG. 32 (SEQ ID NO: 32), FIG. 34 (SEQ ID NO: 34), FIG. 36 (SEQ ID NO:36), FIG. 38 (SEQ ID NO: 38), FIG. 40 (SEQ ID NO: 40), FIG. 42 (SEQ IDNO: 42), FIG. 44 (SEQ ID NO: 44), FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQID NO: 48), FIG. 50 (SEQ ID NO: 50), FIG. 52 (SEQ ID NO: 52), FIG. 54(SEQ ID NO: 54), FIG. 56 (SEQ ID NO: 56), FIG. 58 (SEQ ID NO: 58), FIG.60 (SEQ ID NO: 60), FIG. 62 (SEQ ID NO: 62), FIG. 64 (SEQ ID NO: 64),FIG. 66 (SEQ ID NO: 66), FIG. 68 (SEQ ID NO: 68), FIG. 70 (SEQ ID NO:70), FIG. 72 (SEQ ID NO: 72), FIG. 74 (SEQ ID NO: 74), FIG. 76 (SEQ IDNO: 76), FIG. 78 (SEQ ID NO: 78), FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQID NO: 82), FIG. 84 (SEQ ID NO: 84), FIG. 86 (SEQ ID NO: 86), FIG. 88(SEQ ID NO: 88), FIG. 90 (SEQ ID NO: 90), FIG. 92 (SEQ ID NO: 92), FIG.94 (SEQ ID NO: 94), FIG. 96 (SEQ ID NO: 96), FIG. 98 (SEQ ID NO: 98),FIG. 100 (SEQ ID NO: 100), FIG. 102 (SEQ ID NO: 102), FIG. 104 (SEQ IDNO: 104), FIG. 106 (SEQ ID NO: 106), FIG. 108 (SEQ ID NO: 108), FIG. 110(SEQ ID NO: 110), FIG. 112 (SEQ ID NO: 112), FIG. 114 (SEQ ID NO: 114),FIG. 116 (SEQ ID NO: 116), FIG. 118 (SEQ ID NO: 118), FIG. 120 (SEQ IDNO: 120), FIG. 122 (SEQ ID NO: 122), FIG. 124 (SEQ ID NO: 124), FIG. 126(SEQ ID NO: 126), FIG. 128 (SEQ ID NO: 128), FIG. 130 (SEQ ID NO: 130),FIG. 132 (SEQ ID NO: 132), FIG. 134 (SEQ ID NO: 134), FIG. 136 (SEQ IDNO: 136), FIG. 138 (SEQ ID NO: 138), FIG. 140 (SEQ ID NO: 140), FIG. 142(SEQ ID NO: 142), FIG. 144 (SEQ ID NO: 144), FIG. 146 (SEQ ID NO: 146),FIG. 148 (SEQ ID NO: 148), FIG. 150 (SEQ ID NO: 150), FIG. 152 (SEQ IDNO: 152), FIG. 154 (SEQ ID NO: 154), FIG. 156 (SEQ ID NO: 156), FIG. 158(SEQ ID NO: 158), FIG. 160 (SEQ ID NO: 160), FIG. 162 (SEQ ID NO: 162),FIG. 164 (SEQ ID NO: 164), FIG. 166 (SEQ ID NO: 166), FIG. 168 (SEQ IDNO: 168), FIG. 170 (SEQ ID NO: 170), FIG. 172 (SEQ ID NO: 172), FIG. 174(SEQ ID NO: 174), FIG. 176 (SEQ ID NO: 176), FIG. 178 (SEQ ID NO: 178),FIG. 180 (SEQ ID NO: 180), FIG. 182 (SEQ ID NO: 182), FIG. 184 (SEQ IDNO: 184), FIG. 186 (SEQ ID NO: 186), FIG. 188 (SEQ ID NO: 188), FIG. 190(SEQ ID NO: 190), FIG. 192 (SEQ ID NO: 192), FIG. 194 (SEQ ID NO: 194),FIG. 196 (SEQ ID NO: 196), FIG. 198 (SEQ ID NO: 198), FIG. 200 (SEQ IDNO: 200), FIG. 202 (SEQ ID NO: 202), FIG. 204 (SEQ ID NO: 204), FIG. 206(SEQ ID NO: 206), FIG. 208 (SEQ ID NO: 208), FIG. 210 (SEQ ID NO: 210),FIG. 212 (SEQ ID NO: 212), FIG. 214 (SEQ ID NO: 214), FIG. 216 (SEQ IDNO: 216), FIG. 218 (SEQ ID NO: 218), FIG. 220 (SEQ ID NO: 220), FIG. 222(SEQ ID NO: 222), FIG. 224 (SEQ ID NO: 224), FIG. 226 (SEQ ID NO: 226),FIG. 228 (SEQ ID NO: 228), FIG. 230 (SEQ ID NO: 230), FIG. 232 (SEQ IDNO: 232), FIG. 234 (SEQ ID NO: 234), FIG. 236 (SEQ ID NO: 236), FIG. 238(SEQ ID NO: 238), FIG. 240 (SEQ ID NO: 240), FIG. 242 (SEQ ID NO: 242),FIG. 244 (SEQ ID NO: 244), FIG. 246 (SEQ ID NO: 246), FIG. 248 (SEQ IDNO: 248), FIG. 250 (SEQ ID NO: 250), FIG. 252 (SEQ ID NO: 252), FIG. 254(SEQ ID NO: 254), FIG. 256 (SEQ ID NO: 256), FIG. 258 (SEQ ID NO: 258),FIG. 260 (SEQ ID NO: 260), FIG. 262 (SEQ ID NO: 262), FIG. 264 (SEQ IDNO: 264), FIG. 266 (SEQ ID NO: 266), FIG. 268 (SEQ ID NO: 268), FIG. 270(SEQ ID NO: 270), FIG. 272 (SEQ ID NO: 272), FIG. 274 (SEQ ID NO: 274),FIG. 276 (SEQ ID NO: 276), FIG. 278 (SEQ ID NO: 278), FIG. 280 (SEQ IDNO: 280), FIG. 282 (SEQ ID NO: 282), FIG. 284 (SEQ ID NO: 284), FIG. 286(SEQ ID NO: 286), FIG. 288 (SEQ ID NO: 288), FIG. 290 (SEQ ID NO: 290),FIG. 292 (SEQ ID NO: 292), FIG. 294 (SEQ ID NO: 294), FIG. 296 (SEQ IDNO: 296), FIG. 298 (SEQ ID NO: 298), FIG. 300 (SEQ ID NO: 300), FIG. 302(SEQ ID NO: 302), FIG. 304 (SEQ ID NO: 304), FIG. 306 (SEQ ID NO: 306),FIG. 308 (SEQ ID NO: 308), FIG. 310 (SEQ ID NO: 310), FIG. 312 (SEQ IDNO: 312), FIG. 314 (SEQ ID NO: 314), FIG. 316 (SEQ ID NO: 316), FIG. 318(SEQ ID NO: 318), FIG. 320 (SEQ ID NO: 320), FIG. 322 (SEQ ID NO: 322),FIG. 324 (SEQ ID NO: 324), FIG. 326 (SEQ ID NO: 326), FIG. 328 (SEQ IDNO: 328), FIG. 330 (SEQ ID NO: 330), FIG. 332 (SEQ ID NO: 332), FIG. 334(SEQ ID NO: 334), FIG. 336 (SEQ ID NO: 336), FIG. 338 (SEQ ID NO: 338),FIG. 340 (SEQ ID NO: 340), FIG. 342 (SEQ ID NO: 342), FIG. 344 (SEQ IDNO: 344), FIG. 346 (SEQ ID NO: 346), FIG. 348 (SEQ ID NO: 348), FIG. 350(SEQ ID NO: 350), FIG. 352 (SEQ ID NO: 352), FIG. 354 (SEQ ID NO: 354),FIG. 356 (SEQ ID NO: 356), FIG. 358 (SEQ ID NO: 358), FIG. 360 (SEQ IDNO: 360), FIG. 362 (SEQ ID NO: 362), FIG. 364 (SEQ ID NO: 364), FIG. 366(SEQ ID NO: 366), FIG. 368 (SEQ ID NO: 368), FIG. 370 (SEQ ID NO: 370),FIG. 372 (SEQ ID NO: 372) or FIG. 374 (SEQ ID NO: 374), lacking itsassociated signal peptide; (b) an amino acid sequence of anextracellular domain of the polypeptide shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4), FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ ID NO: 8),FIG. 10 (SEQ ID NO: 10), FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQ ID NO:14), FIG. 16 (SEQ ID NO: 16), FIG. 18 (SEQ ID NO: 18), FIG. 20 (SEQ IDNO: 20), FIG. 22 (SEQ ID NO: 22), FIG. 24 (SEQ ID NO: 24), FIG. 26 (SEQID NO: 26), FIG. 28 (SEQ ID NO: 28), FIG. 30 (SEQ ID NO: 30), FIG. 32(SEQ ID NO: 32), FIG. 34 (SEQ ID NO: 34), FIG. 36 (SEQ ID NO: 36), FIG.38 (SEQ ID NO: 38), FIG. 40 (SEQ ID NO: 40), FIG. 42 (SEQ ID NO: 42),FIG. 44 (SEQ ID NO: 44), FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQ ID NO:48), FIG. 50 (SEQ ID NO: 50), FIG. 52 (SEQ ID NO: 52), FIG. 54 (SEQ IDNO: 54), FIG. 56 (SEQ ID NO: 56), FIG. 58 (SEQ ID NO: 58), FIG. 60 (SEQID NO: 60), FIG. 62 (SEQ ID NO: 62), FIG. 64 (SEQ ID NO: 64), FIG. 66(SEQ ID NO: 66), FIG. 68 (SEQ ID NO: 68), FIG. 70 (SEQ ID NO: 70), FIG.72 (SEQ ID NO: 72), FIG. 74 (SEQ ID NO: 74), FIG. 76 (SEQ ID NO: 76),FIG. 78 (SEQ ID NO: 78), FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQ ID NO:82), FIG. 84 (SEQ ID NO: 84), FIG. 86 (SEQ ID NO: 86), FIG. 88 (SEQ IDNO: 88), FIG. 90 (SEQ ID NO: 90), FIG. 92 (SEQ ID NO: 92), FIG. 94 (SEQID NO: 94), FIG. 96 (SEQ ID NO: 96), FIG. 98 (SEQ ID NO: 98), FIG. 100(SEQ ID NO: 100), FIG. 102 (SEQ ID NO: 102), FIG. 104 (SEQ ID NO: 104),FIG. 106 (SEQ ID NO: 106), FIG. 108 (SEQ ID NO: 108), FIG. 110 (SEQ IDNO: 110), FIG. 112 (SEQ ID NO: 112), FIG. 114 (SEQ ID NO: 114), FIG. 116(SEQ ID NO: 116), FIG. 118 (SEQ ID NO: 118), FIG. 120 (SEQ ID NO: 120),FIG. 122 (SEQ ID NO: 122), FIG. 124 (SEQ ID NO: 124), FIG. 126 (SEQ IDNO: 126), FIG. 128 (SEQ ID NO: 128), FIG. 130 (SEQ ID NO: 130), FIG. 132(SEQ ID NO: 132), FIG. 134 (SEQ ID NO: 134), FIG. 136 (SEQ ID NO: 136),FIG. 138 (SEQ ID NO: 138), FIG. 140 (SEQ ID NO: 140), FIG. 142 (SEQ IDNO: 142), FIG. 144 (SEQ ID NO: 144), FIG. 146 (SEQ ID NO: 146), FIG. 148(SEQ ID NO: 148), FIG. 150 (SEQ ID NO: 150), FIG. 152 (SEQ ID NO: 152),FIG. 154 (SEQ ID NO: 154), FIG. 156 (SEQ ID NO: 156), FIG. 158 (SEQ IDNO: 158), FIG. 160 (SEQ ID NO: 160), FIG. 162 (SEQ ID NO: 162), FIG. 164(SEQ ID NO: 164), FIG. 166 (SEQ ID NO: 166), FIG. 168 (SEQ ID NO: 168),FIG. 170 (SEQ ID NO: 170), FIG. 172 (SEQ ID NO: 172), FIG. 174 (SEQ IDNO: 174), FIG. 176 (SEQ ID NO: 176), FIG. 178 (SEQ ID NO: 178), FIG. 180(SEQ ID NO: 180), FIG. 182 (SEQ ID NO: 182), FIG. 184 (SEQ ID NO: 184),FIG. 186 (SEQ ID NO: 186), FIG. 188 (SEQ ID NO: 188), FIG. 190 (SEQ IDNO: 190), FIG. 192 (SEQ ID NO: 192), FIG. 194 (SEQ ID NO: 194), FIG. 196(SEQ ID NO: 196), FIG. 198 (SEQ ID NO: 198), FIG. 200 (SEQ ID NO: 200),FIG. 202 (SEQ ID NO: 202), FIG. 204 (SEQ ID NO: 204), FIG. 206 (SEQ IDNO: 206), FIG. 208 (SEQ ID NO: 208), FIG. 210 (SEQ ID NO: 210), FIG. 212(SEQ ID NO: 212), FIG. 214 (SEQ ID NO: 214), FIG. 216 (SEQ ID NO: 216),FIG. 218 (SEQ ID NO: 218), FIG. 220 (SEQ ID NO: 220), FIG. 222 (SEQ IDNO: 222), FIG. 224 (SEQ ID NO: 224), FIG. 226 (SEQ ID NO: 226), FIG. 228(SEQ ID NO: 228), FIG. 230 (SEQ ID NO: 230), FIG. 232 (SEQ ID NO: 232),FIG. 234 (SEQ ID NO: 234), FIG. 236 (SEQ ID NO: 236), FIG. 238 (SEQ IDNO: 238), FIG. 240 (SEQ ID NO: 240), FIG. 242 (SEQ ID NO: 242), FIG. 244(SEQ ID NO: 244), FIG. 246 (SEQ ID NO: 246), FIG. 248 (SEQ ID NO: 248),FIG. 250 (SEQ ID NO: 250), FIG. 252 (SEQ ID NO: 252), FIG. 254 (SEQ IDNO: 254), FIG. 256 (SEQ ID NO: 256), FIG. 258 (SEQ ID NO: 258), FIG. 260(SEQ ID NO: 260), FIG. 262 (SEQ ID NO: 262), FIG. 264 (SEQ ID NO: 264),FIG. 266 (SEQ ID NO: 266), FIG. 268 (SEQ ID NO: 268), FIG. 270 (SEQ IDNO: 270), FIG. 272 (SEQ ID NO: 272), FIG. 274 (SEQ ID NO: 274), FIG. 276(SEQ ID NO: 276), FIG. 278 (SEQ ID NO: 278), FIG. 280 (SEQ ID NO: 280),FIG. 282 (SEQ ID NO: 282), FIG. 284 (SEQ ID NO: 284), FIG. 286 (SEQ IDNO: 286), FIG. 288 (SEQ ID NO: 288), FIG. 290 (SEQ ID NO: 290), FIG. 292(SEQ ID NO: 292), FIG. 294 (SEQ ID NO: 294), FIG. 296 (SEQ ID NO: 296),FIG. 298 (SEQ ID NO: 298), FIG. 300 (SEQ ID NO: 300), FIG. 302 (SEQ IDNO: 302), FIG. 304 (SEQ ID NO: 304), FIG. 306 (SEQ ID NO: 306), FIG. 308(SEQ ID NO: 308), FIG. 310 (SEQ ID NO: 310), FIG. 312 (SEQ ID NO: 312),FIG. 314 (SEQ ID NO: 314), FIG. 316 (SEQ ID NO: 316), FIG. 318 (SEQ IDNO: 318), FIG. 320 (SEQ ID NO: 320), FIG. 322 (SEQ ID NO: 322), FIG. 324(SEQ ID NO: 324), FIG. 326 (SEQ ID NO: 326), FIG. 328 (SEQ ID NO: 328),FIG. 330 (SEQ ID NO: 330), FIG. 332 (SEQ ID NO: 332), FIG. 334 (SEQ IDNO: 334), FIG. 336 (SEQ ID NO: 336), FIG. 338 (SEQ ID NO: 338), FIG. 340(SEQ ID NO: 340), FIG. 342 (SEQ ID NO: 342), FIG. 344 (SEQ ID NO: 344),FIG. 346 (SEQ ID NO: 346), FIG. 348 (SEQ ID NO: 348), FIG. 350 (SEQ IDNO: 350), FIG. 352 (SEQ ID NO: 352), FIG. 354 (SEQ ID NO: 354), FIG. 356(SEQ ID NO: 356), FIG. 358 (SEQ ID NO: 358), FIG. 360 (SEQ ID NO: 360),FIG. 362 (SEQ ID NO: 362), FIG. 364 (SEQ ID NO: 364), FIG. 366 (SEQ IDNO: 366), FIG. 368 (SEQ ID NO: 368), FIG. 370 (SEQ ID NO: 370), FIG. 372(SEQ ID NO: 372) or FIG. 374 (SEQ ID NO: 374), with its associatedsignal peptide; or (c) an amino acid sequence of an extracellular domainof the polypeptide shown in FIG. 2 (SEQ ID NO: 2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ ID NO: 8), FIG. 10 (SEQ ID NO:10), FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQ ID NO: 14), FIG. 16 (SEQ IDNO: 16), FIG. 18 (SEQ ID NO: 18), FIG. 20 (SEQ ID NO: 20), FIG. 22 (SEQID NO: 22), FIG. 24 (SEQ ID NO: 24), FIG. 26 (SEQ ID NO: 26), FIG. 28(SEQ ID NO: 28), FIG. 30 (SEQ ID NO: 30), FIG. 32 (SEQ ID NO: 32), FIG.34 (SEQ ID NO: 34), FIG. 36 (SEQ ID NO: 36), FIG. 38 (SEQ ID NO: 38),FIG. 40 (SEQ ID NO: 40), FIG. 42 (SEQ ID NO: 42), FIG. 44 (SEQ ID NO:44), FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQ ID NO: 48), FIG. 50 (SEQ IDNO: 50), FIG. 52 (SEQ ID NO: 52), FIG. 54 (SEQ ID NO: 54), FIG. 56 (SEQID NO: 56), FIG. 58 (SEQ ID NO: 58), FIG. 60 (SEQ ID NO: 60), FIG. 62(SEQ ID NO: 62), FIG. 64 (SEQ ID NO: 64), FIG. 66 (SEQ ID NO: 66), FIG.68 (SEQ ID NO: 68), FIG. 70 (SEQ ID NO: 70), FIG. 72 (SEQ ID NO: 72),FIG. 74 (SEQ ID NO: 74), FIG. 76 (SEQ ID NO: 76), FIG. 78 (SEQ ID NO:78), FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQ ID NO: 82), FIG. 84 (SEQ IDNO: 84), FIG. 86 (SEQ ID NO: 86), FIG. 88 (SEQ ID NO: 88), FIG. 90 (SEQID NO: 90), FIG. 92 (SEQ ID NO: 92), FIG. 94 (SEQ ID NO: 94), FIG. 96(SEQ ID NO: 96), FIG. 98 (SEQ ID NO: 98), FIG. 100 (SEQ ID NO: 100),FIG. 102 (SEQ ID NO: 102), FIG. 104 (SEQ ID NO: 104), FIG. 106 (SEQ IDNO: 106), FIG. 108 (SEQ ID NO: 108), FIG. 110 (SEQ ID NO: 110), FIG. 112(SEQ ID NO: 112), FIG. 114 (SEQ ID NO: 114), FIG. 116 (SEQ ID NO: 116),FIG. 118 (SEQ ID NO: 118), FIG. 120 (SEQ ID NO: 120), FIG. 122 (SEQ IDNO: 122), FIG. 124 (SEQ ID NO: 124), FIG. 126 (SEQ ID NO: 126), FIG. 128(SEQ ID NO: 128), FIG. 130 (SEQ ID NO: 130), FIG. 132 (SEQ ID NO: 132),FIG. 134 (SEQ ID NO: 134), FIG. 136 (SEQ ID NO: 136), FIG. 138 (SEQ IDNO: 138), FIG. 140 (SEQ ID NO: 140), FIG. 142 (SEQ ID NO: 142), FIG. 144(SEQ ID NO: 144), FIG. 146 (SEQ ID NO: 146), FIG. 148 (SEQ ID NO: 148),FIG. 150 (SEQ ID NO: 150), FIG. 152 (SEQ ID NO: 152), FIG. 154 (SEQ IDNO: 154), FIG. 156 (SEQ ID NO: 156), FIG. 158 (SEQ ID NO: 158), FIG. 160(SEQ ID NO: 160), FIG. 162 (SEQ ID NO: 162), FIG. 164 (SEQ ID NO: 164),FIG. 166 (SEQ ID NO: 166), FIG. 168 (SEQ ID NO: 168), FIG. 170 (SEQ IDNO: 170), FIG. 172 (SEQ ID NO: 172), FIG. 174 (SEQ ID NO: 174), FIG. 176(SEQ ID NO: 176), FIG. 178 (SEQ ID NO: 178), FIG. 180 (SEQ ID NO: 180),FIG. 182 (SEQ ID NO: 182), FIG. 184 (SEQ ID NO: 184), FIG. 186 (SEQ IDNO: 186), FIG. 188 (SEQ ID NO: 188), FIG. 190 (SEQ ID NO: 190), FIG. 192(SEQ ID NO: 192), FIG. 194 (SEQ ID NO: 194), FIG. 196 (SEQ ID NO: 196),FIG. 198 (SEQ ID NO: 198), FIG. 200 (SEQ ID NO: 200), FIG. 202 (SEQ IDNO: 202), FIG. 204 (SEQ ID NO: 204), FIG. 206 (SEQ ID NO: 206), FIG. 208(SEQ ID NO: 208), FIG. 210 (SEQ ID NO: 210), FIG. 212 (SEQ ID NO: 212),FIG. 214 (SEQ ID NO: 214), FIG. 216 (SEQ ID NO: 216), FIG. 218 (SEQ IDNO: 218), FIG. 220 (SEQ ID NO: 220), FIG. 222 (SEQ ID NO: 222), FIG. 224(SEQ ID NO: 224), FIG. 226 (SEQ ID NO: 226), FIG. 228 (SEQ ID NO: 228),FIG. 230 (SEQ ID NO: 230), FIG. 232 (SEQ ID NO: 232), FIG. 234 (SEQ IDNO: 234), FIG. 236 (SEQ ID NO: 236), FIG. 238 (SEQ ID NO: 238), FIG. 240(SEQ ID NO: 240), FIG. 242 (SEQ ID NO: 242), FIG. 244 (SEQ ID NO: 244),FIG. 246 (SEQ ID NO: 246), FIG. 248 (SEQ ID NO: 248), FIG. 250 (SEQ IDNO: 250), FIG. 252 (SEQ ID NO: 252), FIG. 254 (SEQ ID NO: 254), FIG. 256(SEQ ID NO: 256), FIG. 258 (SEQ ID NO: 258), FIG. 260 (SEQ ID NO: 260),FIG. 262 (SEQ ID NO: 262), FIG. 264 (SEQ ID NO: 264), FIG. 266 (SEQ IDNO: 266), FIG. 268 (SEQ ID NO: 268), FIG. 270 (SEQ ID NO: 270), FIG. 272(SEQ ID NO: 272), FIG. 274 (SEQ ID NO: 274), FIG. 276 (SEQ ID NO: 276),FIG. 278 (SEQ ID NO: 278), FIG. 280 (SEQ ID NO: 280), FIG. 282 (SEQ IDNO: 282), FIG. 284 (SEQ ID NO: 284), FIG. 286 (SEQ ID NO: 286), FIG. 288(SEQ ID NO: 288), FIG. 290 (SEQ ID NO: 290), FIG. 292 (SEQ ID NO: 292),FIG. 294 (SEQ ID NO: 294), FIG. 296 (SEQ ID NO: 296), FIG. 298 (SEQ IDNO: 298), FIG. 300 (SEQ ID NO: 300), FIG. 302 (SEQ ID NO: 302), FIG. 304(SEQ ID NO: 304), FIG. 306 (SEQ ID NO: 306), FIG. 308 (SEQ ID NO: 308),FIG. 310 (SEQ ID NO: 310), FIG. 312 (SEQ ID NO: 312), FIG. 314 (SEQ IDNO: 314), FIG. 316 (SEQ ID NO: 316), FIG. 318 (SEQ ID NO: 318), FIG. 320(SEQ ID NO: 320), FIG. 322 (SEQ ID NO: 322), FIG. 324 (SEQ ID NO: 324),FIG. 326 (SEQ ID NO: 326), FIG. 328 (SEQ ID NO: 328), FIG. 330 (SEQ IDNO: 330), FIG. 332 (SEQ ID NO: 332), FIG. 334 (SEQ ID NO: 334), FIG. 336(SEQ ID NO: 336), FIG. 338 (SEQ ID NO: 338), FIG. 340 (SEQ ID NO: 340),FIG. 342 (SEQ ID NO: 342), FIG. 344 (SEQ ID NO: 344), FIG. 346 (SEQ IDNO: 346), FIG. 348 (SEQ ID NO: 348), FIG. 350 (SEQ ID NO: 350), FIG. 352(SEQ ID NO: 352), FIG. 354 (SEQ ID NO: 354), FIG. 356 (SEQ ID NO: 356),FIG. 358 (SEQ ID NO: 358), FIG. 360 (SEQ ID NO: 360), FIG. 362 (SEQ IDNO: 362), FIG. 364 (SEQ ID NO: 364), FIG. 366 (SEQ ID NO: 366), FIG. 368(SEQ ID NO: 368), FIG. 370 (SEQ ID NO: 370), FIG. 372 (SEQ ID NO: 372)or FIG. 374 (SEQ ID NO: 374), lacking its associated signal peptide. 20.A method for treating a cardiovascular, endothelial or anglogenicdisorder in a mammal comprising administering to the mammal atherapeutically effective amount of a polypeptide shown in FIG. 2 (SEQID NO: 2), FIG. 4 (SEQ ID NO: 4), FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ IDNO: 8), FIG. 10 (SEQ ID NO: 10), FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQID NO: 14), FIG. 16 (SEQ ID NO: 16), FIG. 18 (SEQ ID NO: 18), FIG. 20(SEQ ID NO: 20), FIG. 22 (SEQ ID NO: 22), FIG. 24 (SEQ ID NO: 24), FIG.26 (SEQ ID NO: 26), FIG. 28 (SEQ ID NO: 28), FIG. 30 (SEQ ID NO: 30),FIG. 32 (SEQ ID NO: 32), FIG. 34 (SEQ ID NO: 34), FIG. 36 (SEQ ID NO:36), FIG. 38 (SEQ ID NO: 38), FIG. 40 (SEQ ID NO: 40), FIG. 42 (SEQ IDNO: 42), FIG. 44 (SEQ ID NO: 44), FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQID NO: 48), FIG. 50 (SEQ ID NO: 50), FIG. 52 (SEQ ID NO: 52), FIG. 54(SEQ ID NO: 54), FIG. 56 (SEQ ID NO: 56), FIG. 58 (SEQ ID NO: 58), FIG.60 (SEQ ID NO: 60), FIG. 62 (SEQ ID NO: 62), FIG. 64 (SEQ ID NO: 64),FIG. 66 (SEQ ID NO: 66), FIG. 68 (SEQ ID NO: 68), FIG. 70 (SEQ ID NO:70), FIG. 72 (SEQ ID NO: 72), FIG. 74 (SEQ ID NO: 74), FIG. 76 (SEQ IDNO: 76), FIG. 78 (SEQ ID NO: 78), FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQID NO: 82), FIG. 84 (SEQ ID NO: 84), FIG. 86 (SEQ ID NO: 86), FIG. 88(SEQ ID NO: 88), FIG. 90 (SEQ ID NO: 90), FIG. 92 (SEQ ID NO: 92), FIG.94 (SEQ ID NO: 94), FIG. 96 (SEQ ID NO: 96), FIG. 98 (SEQ ID NO: 98),FIG. 100 (SEQ ID NO: 100), FIG. 102 (SEQ ID NO: 102), FIG. 104 (SEQ IDNO: 104), FIG. 106 (SEQ ID NO: 106), FIG. 108 (SEQ ID NO: 108), FIG. 110(SEQ ID NO: 110), FIG. 112 (SEQ ID NO: 112), FIG. 114 (SEQ ID NO: 114),FIG. 116 (SEQ ID NO: 116), FIG. 118 (SEQ ID NO: 118), FIG. 120 (SEQ IDNO: 120), FIG. 122 (SEQ ID NO: 122), FIG. 124 (SEQ ID NO: 124), FIG. 126(SEQ ID NO: 126), FIG. 128 (SEQ ID NO: 128), FIG. 130 (SEQ ID NO: 130),FIG. 132 (SEQ ID NO: 132), FIG. 134 (SEQ ID NO: 134), FIG. 136 (SEQ IDNO: 136), FIG. 138 (SEQ ID NO: 138), FIG. 140 (SEQ ID NO: 140), FIG. 142(SEQ ID NO: 142), FIG. 144 (SEQ ID NO: 144), FIG. 146 (SEQ ID NO: 146),FIG. 148 (SEQ ID NO: 148), FIG. 150 (SEQ ID NO: 150), FIG. 152 (SEQ IDNO: 152), FIG. 154 (SEQ ID NO: 154), FIG. 156 (SEQ ID NO: 156), FIG. 158(SEQ ID NO: 158), FIG. 160 (SEQ ID NO: 160), FIG. 162 (SEQ ID NO: 162),FIG. 164 (SEQ ID NO: 164), FIG. 166 (SEQ ID NO: 166), FIG. 168 (SEQ IDNO: 168), FIG. 170 (SEQ ID NO: 170), FIG. 172 (SEQ ID NO: 172), FIG. 174(SEQ ID NO: 174), FIG. 176 (SEQ ID NO: 176), FIG. 178 (SEQ ID NO: 178),FIG. 180 (SEQ ID NO: 180), FIG. 182 (SEQ ID NO: 182), FIG. 184 (SEQ IDNO: 184), FIG. 186 (SEQ ID NO: 186), FIG. 188 (SEQ ID NO: 188), FIG. 190(SEQ ID NO: 190), FIG. 192 (SEQ ID NO: 192), FIG. 194 (SEQ ID NO: 194),FIG. 196 (SEQ ID NO: 196), FIG. 198 (SEQ ID NO: 198), FIG. 200 (SEQ IDNO: 200), FIG. 202 (SEQ ID NO: 202), FIG. 204 (SEQ ID NO: 204), FIG. 206(SEQ ID NO: 206), FIG. 208 (SEQ ID NO: 208), FIG. 210 (SEQ ID NO: 210),FIG. 212 (SEQ ID NO: 212), FIG. 214 (SEQ ID NO: 214), FIG. 216 (SEQ IDNO: 216), FIG. 218 (SEQ ID NO: 218), FIG. 220 (SEQ ID NO: 220), FIG. 222(SEQ ID NO: 222), FIG. 224 (SEQ ID NO: 224), FIG. 226 (SEQ ID NO: 226),FIG. 228 (SEQ ID NO: 228), FIG. 230 (SEQ ID NO: 230), FIG. 232 (SEQ IDNO: 232), FIG. 234 (SEQ ID NO: 234), FIG. 236 (SEQ ID NO: 236), FIG. 238(SEQ ID NO: 238), FIG. 240 (SEQ ID NO: 240), FIG. 242 (SEQ ID NO: 242),FIG. 244 (SEQ ID NO: 244), FIG. 246 (SEQ ID NO: 246), FIG. 248 (SEQ IDNO: 248), FIG. 250 (SEQ ID NO: 250), FIG. 252 (SEQ ID NO: 252), FIG. 254(SEQ ID NO: 254), FIG. 256 (SEQ ID NO: 256), FIG. 258 (SEQ ID NO: 258),FIG. 260 (SEQ ID NO: 260), FIG. 262 (SEQ ID NO: 262), FIG. 264 (SEQ IDNO: 264), FIG. 266 (SEQ ID NO: 266), FIG. 268 (SEQ ID NO: 268), FIG. 270(SEQ ID NO: 270), FIG. 272 (SEQ ID NO: 272), FIG. 274 (SEQ ID NO: 274),FIG. 276 (SEQ ID NO: 276), FIG. 278 (SEQ ID NO: 278), FIG. 280 (SEQ IDNO: 280), FIG. 282 (SEQ ID NO: 282), FIG. 284 (SEQ ID NO: 284), FIG. 286(SEQ ID NO: 286), FIG. 288 (SEQ ID NO: 288), FIG. 290 (SEQ ID NO: 290),FIG. 292 (SEQ ID NO: 292), FIG. 294 (SEQ ID NO: 294), FIG. 296 (SEQ IDNO: 296), FIG. 298 (SEQ ID NO: 298), FIG. 300 (SEQ ID NO: 300), FIG. 302(SEQ ID NO: 302), FIG. 304 (SEQ ID NO: 304), FIG. 306 (SEQ ID NO: 306),FIG. 308 (SEQ ID NO: 308), FIG. 310 (SEQ ID NO: 310), FIG. 312 (SEQ IDNO: 312), FIG. 314 (SEQ ID NO: 314), FIG. 316 (SEQ ID NO: 316), FIG. 318(SEQ ID NO: 318), FIG. 320 (SEQ ID NO: 320), FIG. 322 (SEQ ID NO: 322),FIG. 324 (SEQ ID NO: 324), FIG. 326 (SEQ ID NO: 326), FIG. 328 (SEQ IDNO: 328), FIG. 330 (SEQ ID NO: 330), FIG. 332 (SEQ ID NO: 332), FIG. 334(SEQ ID NO: 334), FIG. 336 (SEQ ID NO: 336), FIG. 338 (SEQ ID NO: 338),FIG. 340 (SEQ ID NO: 340), FIG. 342 (SEQ ID NO: 342), FIG. 344 (SEQ IDNO: 344), FIG. 346 (SEQ ID NO: 346), FIG. 348 (SEQ ID NO: 348), FIG. 350(SEQ ID NO: 350), FIG. 352 (SEQ ID NO: 352), FIG. 354 (SEQ ID NO: 354),FIG. 356 (SEQ ID NO: 356), FIG. 358 (SEQ ID NO: 358), FIG. 360 (SEQ IDNO: 360), FIG. 362 (SEQ ID NO: 362), FIG. 364 (SEQ ID NO: 364), FIG. 366(SEQ ID NO: 366), FIG. 368 (SEQ ID NO: 368), FIG. 370 (SEQ ID NO: 370),FIG. 372 (SEQ ID NO: 372) or FIG. 374 (SEQ ID NO: 374), or agonist orantagonist thereof.
 21. The method according to claim 20, wherein themammal is human.
 22. The method of claim 21, wherein the human hassuffered myocardial infarction.
 23. The method of claim 21, wherein thehuman has cardiac hypertrophy, trauma, a cancer, or age-related maculardegeneration.
 24. The method of claim 23, wherein the cardiachypertrophy is characterized by the presence of an elevated level ofPGF_(2α).
 25. The method of claim 20, wherein the polypeptide isadministered together with a cardiovascular, endothelial or angiogenicagent.
 26. The method of claim 23, wherein the polypeptide isadministered following primary angioplasty.
 27. The method of claim 20,wherein the cardiovascular, endothelial or angiogenic disorder iscancer.
 28. The method of claim 27, wherein the polypeptide isadministered in combination with a chemotherapeutic agent, a growthinhibitory agent or a cytotoxic agent.
 29. The method of claim 20,wherein said agonist is an antibody to said polypeptide.
 30. The methodof claim 20, wherein said antagonist is an antibody to said polypeptide.31. A method for treating a cardiovascular, endothelial or angiogenicdisorder in a mammal comprising administering to the mammal a nucleicacid molecule that encodes a polypeptide shown in FIG. 2 (SEQ ID NO: 2),FIG. 4 (SEQ ID NO: 4), FIG. 6 (SEQ ID NO: 6), FIG. 8 (SEQ ID NO: 8),FIG. 10 (SEQ ID NO: 10), FIG. 12 (SEQ ID NO: 12), FIG. 14 (SEQ ID NO:14), FIG. 16 (SEQ ID NO: 16), FIG. 18 (SEQ ID NO: 18), FIG. 20 (SEQ IDNO: 20), FIG. 22 (SEQ ID NO: 22), FIG. 24 (SEQ ID NO: 24), FIG. 26 (SEQID NO: 26), FIG. 28 (SEQ ID NO: 28), FIG. 30 (SEQ ID NO: 30), FIG. 32(SEQ ID NO: 32), FIG. 34 (SEQ ID NO: 34), FIG. 36 (SEQ ID NO: 36), FIG.38 (SEQ ID NO: 38), FIG. 40 (SEQ ID NO: 40), FIG. 42 (SEQ ID NO: 42),FIG. 44 (SEQ ID NO: 44), FIG. 46 (SEQ ID NO: 46), FIG. 48 (SEQ ID NO:48), FIG. 50 (SEQ ID NO: 50), FIG. 52 (SEQ ID NO: 52), FIG. 54 (SEQ IDNO: 54), FIG. 56 (SEQ ID NO: 56), FIG. 58 (SEQ ID NO: 58), FIG. 60 (SEQID NO: 60), FIG. 62 (SEQ ID NO: 62), FIG. 64 (SEQ ID NO: 64), FIG. 66(SEQ ID NO: 66), FIG. 68 (SEQ ID NO: 68), FIG. 70 (SEQ ID NO: 70), FIG.72 (SEQ ID NO: 72), FIG. 74 (SEQ ID NO: 74), FIG. 76 (SEQ ID NO: 76),FIG. 78 (SEQ ID NO: 78), FIG. 80 (SEQ ID NO: 80), FIG. 82 (SEQ ID NO:82), FIG. 84 (SEQ ID NO: 84), FIG. 86 (SEQ ID NO: 86), FIG. 88 (SEQ IDNO: 88), FIG. 90 (SEQ ID NO: 90), FIG. 92 (SEQ ID NO: 92), FIG. 94 (SEQID NO: 94), FIG. 96 (SEQ ID NO: 96), FIG. 98 (SEQ ID NO: 98), FIG. 100(SEQ ID NO: 100), FIG. 102 (SEQ ID NO: 102), FIG. 104 (SEQ ID NO: 104),FIG. 106 (SEQ ID NO: 106), FIG. 108 (SEQ ID NO: 108), FIG. 110 (SEQ IDNO: 110), FIG. 112 (SEQ ID NO: 112), FIG. 114 (SEQ ID NO: 114), FIG. 116(SEQ ID NO: 116), FIG. 118 (SEQ ID NO: 118), FIG. 120 (SEQ ID NO: 120),FIG. 122 (SEQ ID NO: 122), FIG. 124 (SEQ ID NO: 124), FIG. 126 (SEQ IDNO: 126), FIG. 128 (SEQ ID NO: 128), FIG. 130 (SEQ ID NO: 130), FIG. 132(SEQ ID NO: 132), FIG. 134 (SEQ ID NO: 134), FIG. 136 (SEQ ID NO: 136),FIG. 138 (SEQ ID NO: 138), FIG. 140 (SEQ ID NO: 140), FIG. 142 (SEQ IDNO: 142), FIG. 144 (SEQ ID NO: 144), FIG. 146 (SEQ ID NO: 146), FIG. 148(SEQ ID NO: 148), FIG. 150 (SEQ ID NO: 150), FIG. 152 (SEQ ID NO: 152),FIG. 154 (SEQ ID NO: 154), FIG. 156 (SEQ ID NO: 156), FIG. 158 (SEQ IDNO: 158), FIG. 160 (SEQ ID NO: 160), FIG. 162 (SEQ ID NO: 162), FIG. 164(SEQ ID NO: 164), FIG. 166 (SEQ ID NO: 166), FIG. 168 (SEQ ID NO: 168),FIG. 170 (SEQ ID NO: 170), FIG. 172 (SEQ ID NO: 172), FIG. 174 (SEQ IDNO: 174), FIG. 176 (SEQ ID NO: 176), FIG. 178 (SEQ ID NO: 178), FIG. 180(SEQ ID NO: 180), FIG. 182 (SEQ ID NO: 182), FIG. 184 (SEQ ID NO: 184),FIG. 186 (SEQ ID NO: 186), FIG. 188 (SEQ ID NO: 188), FIG. 190 (SEQ IDNO: 190), FIG. 192 (SEQ ID NO: 192), FIG. 194 (SEQ ID NO: 194), FIG. 196(SEQ ID NO: 196), FIG. 198 (SEQ ID NO: 198), FIG. 200 (SEQ ID NO: 200),FIG. 202 (SEQ ID NO: 202), FIG. 204 (SEQ ID NO: 204), FIG. 206 (SEQ IDNO: 206), FIG. 208 (SEQ ID NO: 208), FIG. 210 (SEQ ID NO: 210), FIG. 212(SEQ ID NO: 212), FIG. 214 (SEQ ID NO: 214), FIG. 216 (SEQ ID NO: 216),FIG. 218 (SEQ ID NO: 218), FIG. 220 (SEQ ID NO: 220), FIG. 222 (SEQ IDNO: 222), FIG. 224 (SEQ ID NO: 224), FIG. 226 (SEQ ID NO: 226), FIG. 228(SEQ ID NO: 228), FIG. 230 (SEQ ID NO: 230), FIG. 232 (SEQ ID NO: 232),FIG. 234 (SEQ ID NO: 234), FIG. 236 (SEQ ID NO: 236), FIG. 238 (SEQ IDNO: 238), FIG. 240 (SEQ ID NO: 240), FIG. 242 (SEQ ID NO: 242), FIG. 244(SEQ ID NO: 244), FIG. 246 (SEQ ID NO: 246), FIG. 248 (SEQ ID NO: 248),FIG. 250 (SEQ ID NO: 250), FIG. 252 (SEQ ID NO: 252), FIG. 254 (SEQ IDNO: 254), FIG. 256 (SEQ ID NO: 256), FIG. 258 (SEQ ID NO: 258), FIG. 260(SEQ ID NO: 260), FIG. 262 (SEQ ID NO: 262), FIG. 264 (SEQ ID NO: 264),FIG. 266 (SEQ ID NO: 266), FIG. 268 (SEQ ID NO: 268), FIG. 270 (SEQ IDNO: 270), FIG. 272 (SEQ ID NO: 272), FIG. 274 (SEQ ID NO: 274), FIG. 276(SEQ ID NO: 276), FIG. 278 (SEQ ID NO: 278), FIG. 280 (SEQ ID NO: 280),FIG. 282 (SEQ ID NO: 282), FIG. 284 (SEQ ID NO: 284), FIG. 286 (SEQ IDNO: 286), FIG. 288 (SEQ ID NO: 288), FIG. 290 (SEQ ID NO: 290), FIG. 292(SEQ ID NO: 292), FIG. 294 (SEQ ID NO: 294), FIG. 296 (SEQ ID NO: 296),FIG. 298 (SEQ ID NO: 298), FIG. 300 (SEQ ID NO: 300), FIG. 302 (SEQ IDNO: 302), FIG. 304 (SEQ ID NO: 304), FIG. 306 (SEQ ID NO: 306), FIG. 308(SEQ ID NO: 308), FIG. 310 (SEQ ID NO: 310), FIG. 312 (SEQ ID NO: 312),FIG. 314 (SEQ ID NO: 314), FIG. 316 (SEQ ID NO: 316), FIG. 318 (SEQ IDNO: 318), FIG. 320 (SEQ ID NO: 320), FIG. 322 (SEQ ID NO: 322), FIG. 324(SEQ ID NO: 324), FIG. 326 (SEQ ID NO: 326), FIG. 328 (SEQ ID NO: 328),FIG. 330 (SEQ ID NO: 330), FIG. 332 (SEQ ID NO: 332), FIG. 334 (SEQ IDNO: 334), FIG. 336 (SEQ ID NO: 336), FIG. 338 (SEQ ID NO: 338), FIG. 340(SEQ ID NO: 340), FIG. 342 (SEQ ID NO: 342), FIG. 344 (SEQ ID NO: 344),FIG. 346 (SEQ ID NO: 346), FIG. 348 (SEQ ID NO: 348), FIG. 350 (SEQ IDNO: 350), FIG. 352 (SEQ ID NO: 352), FIG. 354 (SEQ ID NO: 354), FIG. 356(SEQ ID NO: 356), FIG. 358 (SEQ ID NO: 358), FIG. 360 (SEQ ID NO: 360),FIG. 362 (SEQ ID NO: 362), FIG. 364 (SEQ ID NO: 364), FIG. 366 (SEQ IDNO: 366), FIG. 368 (SEQ ID NO: 368), FIG. 370 (SEQ ID NO: 370), FIG. 372(SEQ ID NO: 372) or FIG. 374 (SEQ ID NO: 374), or agonist or antagonistthereof.
 32. The method of claim 31, wherein said agonist is an antibodyto said polypeptide.
 33. The method of claim 31, wherein said antagonistis an antibody to said polypeptide.
 34. The method of claim 31, whereinthe mammal is human.
 35. The method of claim 31, wherein the nucleicacid molecule is administered via ex vivo gene therapy.
 36. A method forinhibiting endothelial cell growth in a mammal comprising administeringto the mammal a PRO229, PRO238, PRO247, PRO444, PRO720, PRO827, PRO1007,PRO1029, PRO1075, PRO1184, PRO1190, PRO1195, PRO1274, PRO1279, PRO1419,PRO1474, PRO1477, PRO1488, PRO1782, PRO1890, PRO4302, PRO4405, PRO5725,PRO5776, PRO6006, PRO7436, PRO9771, PRO10008, PRO21384 or PRO28631polypeptide or agonist thereof, wherein endothelial cell growth in saidmammal is inhibited.
 37. A method for stimulating endothelial cellgrowth in a mammal comprising administering to the mammal a PRO21,PRO181, PRO205, PRO214, PRO221, PRO231, PRO238, PRO241, PRO247, PRO256,PRO258, PRO263, PRO265, PRO295, PRO321, PRO322, PRO337, PRO363, PRO365,PRO533, PRO697, PRO725, PRO771, PRO788, PRO791, PRO819, PRO828, PRO836,PRO846, PRO865, PRO1005, PRO1006, PRO1025, PRO1054, PRO1071, PRO1079,PRO1080, PRO1114, PRO1131, PRO1155, PRO1160, PRO1186, PRO1192, PRO1244,PRO1272, PRO1273, PRO1279, PRO1283, PRO1286, PRO1306, PRO1309, PRO1325,PRO1329, PRO1347, PRO1356, PRO1376, PRO1382, PRO1411, PRO1412, PRO1508,PRO1550, PRO1556, PRO1760, PRO1787, PRO1801, PRO1868, PRO1887, PRO3438,PRO3444, PRO4324, PRO4333, PRO4341, PRO4342, PRO4353, PRO4354, PRO4356,PRO4371, PRO4408, PRO4422, PRO4425, PRO4499, PRO5723, PRO5737, PRO6029,PRO6071, PRO9821, PRO9873, PRO10008, PRO10096, PRO19670, PRO20040,PRO20044, PRO21055 or PRO21384 polypeptide, or agonist thereof, whereinendothelial cell growth in said mammal is stimulated.
 38. A method forinducing cardiac hypertrophy in a mammal comprising administering to themammal a PRO21 polypeptide or agonist thereof, wherein cardiachypertrophy in said mammal is induced.
 39. A method for stimulatingangiogenesis induced by a PRO1376 or PRO1449 polypeptide in a mammalcomprising administering a therapeutically effective amount of saidpolypeptide to the mammal, wherein said angiogenesis is stimulated. 40.A method for inducing endothelial cell apoptosis comprisingadministering to the endothelial cell a PRO4302 polypeptide or agonistthereof, wherein apoptosis in said endothelial cell is induced.
 41. Amethod for stimulating smooth muscle cell growth comprisingadministering to the smooth muscle cell a PRO162, PRO182, PRO204,PRO221, PRO230, PRO256, PRO258, PRO533, PRO697, PRO725, PRO738, PRO826,PRO836, PRO840, PRO846, PRO865, PRO982, PRO1025, PRO1029, PRO1071,PRO1083, PRO1134, PRO1160, PRO1182, PRO1184, PRO1186, PRO1192, PRO1274,PRO1279, PRO1283, PRO1306, PRO1308, PRO1325, PRO1337, PRO1338, PRO1343,PRO1376, PRO1387, PRO1411, PRO1412, PRO1415, PRO1434, PRO1474, PRO1550,PRO1556, PRO1567, PRO1600, PRO1754, PRO1758, PRO1760, PRO1787, PRO1865,PRO1868, PRO1917, PRO1928, PRO3438, PRO3562, PRO4333, PRO4345, PRO4353,PRO4354, PRO4408, PRO4430, PRO4503, PRO6714, PRO9771, PRO9820, PRO9940,PRO10096, PRO21055, PRO21184 or PRO21366 polypeptide, or agonistthereof, wherein smooth muscle cell growth in said smooth muscle cell isstimulated.
 42. A method for inhibiting smooth muscle cell growthcomprising administering to the smooth muscle cell a PRO181, PRO195,PRO1080, PRO1265, PRO1309, PRO1488, PRO4302, PRO4405 or PRO5725polypeptide, or agonist thereof, wherein smooth muscle cell growth insaid smooth muscle cell is stimulated.
 43. A method for inducingendothelial cell tube formation comprising administering to theendothelial cell a PRO178, PRO195, PRO228, PRO301, PRO302, PRO532,PRO724, PRO730, PRO734, PRO793, PRO871, PRO938, PRO1012, PRO1120,PRO1139, PRO1198, PRO1287, PRO1361, PRO1864, PRO1873, PRO2010, PRO3579,PRO4313, PRO4527, PRO4538, PRO4553, PRO4995, PRO5730, PRO6008, PRO7223,PRO7248, PRO7261 polypeptide, or agonist thereof, wherein tube formationin said endothelial cell is induced.